Anti-ly6h antibodies and antibody drug conjugates

ABSTRACT

Disclosed herein are anti-lymphocyte antigen 6 complex, locus H (LY6H) antibodies and antibody drug conjugates (ADCs), including compositions and methods of using said antibodies and ADCs.

RELATED APPLICATIONS

The instant application claims priority to U.S. Provisional ApplicationNo. 62/526,297, filed on Jun. 28, 2017, U.S. Provisional Application No.62/527,172, filed on Jun. 30, 2017, and U.S. Provisional Application No.62/588,520, filed on Nov. 20, 2017, the entire contents of which areexpressly incorporated by reference herein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Jun. 27, 2018, is named127913-00420_SL.txt and is 67,506 bytes in size.

BACKGROUND

LY6H, also known as “lymphocyte antigen 6 complex, locus H,” “LymphocyteAntigen 6H,” “Ly-6H,” and “NMLY6,” is a member of the LY6 family ofglycosylphosphatidylinositol-anchored cell surface glycoproteins thatare expressed on various types of cells. Isolation and characterizationof LY6H was first reported in 1998 (Horie, M, et al. Genomics 53:365-368, 1998).

Human LY6H is synthesized as a 140 amino acid precursor that contains a25 amino acid signal sequence, 20 amino acid propeptide that is removedin the mature form, and a 90 amino acid mature chain. LY6H is highlyexpressed in brain (e.g., cerebral cortex, amygdala, hippocampus andsubthalamic nucleus) and in acute lymphoblastic leukemia cells, such asMOLT-3 and MOLT-4. It is also found in lower levels in testis, pancreas,small intestine and colon. This suggests that LY6H may play a role inboth the central nervous system and the immune system (Horie, M, et al.Genomics 53: 365-368, 1998). Intriguing patterns of expression of Ly6genes on specific subpopulations of lymphoid and myeloid cells suggestthat Ly6 molecules may be involved in the development and homeostasis ofhematopoietic cells (Horie, M, et al. Genomics 53: 365-368, 1998).Further, increased expression of Ly6 family members in multiple cancertype indicate that Ly6 family members may be important targets indeveloping novel cancer therapeutics (Luo, L. et al. Oncotarget 7(10):11165-11193, 2016).

Antibody drug conjugates (ADC) represent a new class of therapeuticscomprising an antibody conjugated to a cytotoxic drug via a chemicallinker. The therapeutic concept of ADCs is to combine bindingcapabilities of an antibody with a drug, where the antibody is used todeliver the drug to a tumor cell by means of binding to a target surfaceantigen.

Accordingly, there remains a need in the art for anti-LY6H antibodiesand ADCs that can be used for therapeutic purposes in the treatment ofcancer.

SUMMARY

In certain aspects, the present invention provides for anti-LY6Hantibodies and antibody drug conjugates (ADCs). In certain embodimentsof the invention, the antibodies, or antigen binding portions thereof,bind to LY6H (SEQ ID NO: 123) or the extracellular domain of LY6H. Inone embodiment, the antibodies, or antigen binding portions thereof, ofthe invention, bind to LY6H with a K_(d) of about 2,000 nM or less,about 1,000 nM or less, about 500 nM or less, about 200 nM or less,about 100 nM or less, about 75 nM or less, about 25 nM or less, about 21nM or less, about 12 nM or less, about 11 nM or less, about 10 nM orless, about 9 nM or less, about 8 nM or less, about 7 nM or less, about6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM orless, about 2 nM or less, about 1 nM or less, about 0.5 nM or less,about 0.3 nM or less, about 0.1 nM or less, or about 0.01 nM or less, orabout 0.001 nM or less.

In yet other embodiments of the invention, anti-LY6H antibody andantibody drug conjugates (ADCs) of the invention (e.g., the LY6Hantibodies of the invention conjugated to a toxin) are capable of beinginternalized. In another embodiment, the anti-LY6H antibody and antibodydrug conjugates (ADCs) of the invention are capable of inducing celldeath of cells endogenously expressing LY6H.

In one aspect of the invention, the present disclosure provides anisolated antibody, or antigen binding portion thereof, that binds tohuman LY6H, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR3 having theamino acid sequence of SEQ ID NO: 4 and a light chain variable regioncomprising a CDR3 having the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the antibody, or antigen binding portion thereof,further comprises a heavy chain variable region comprising a CDR2 havingone of the amino acid sequences selected from SEQ ID NO: 3 and SEQ IDNO: 69 and a light chain variable region comprising a CDR2 having theamino acid sequence of SEQ ID NO: 7. In other embodiments, the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising a CDR1 having the amino acid sequence of SEQ ID NO: 2and a light chain variable region comprising a CDR1 having the aminoacid sequence of either SEQ ID NO: 6.

In one aspect of the invention, the present disclosure provides anisolated antibody, or antigen binding portion thereof, that binds tohuman LY6H, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR3 having theamino acid sequence of SEQ ID NO: 12 and a light chain variable regioncomprising a CDR3 having the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the antibody, or antigen binding portion thereof,further comprises a heavy chain variable region comprising a CDR2 havingthe amino acid sequence of SEQ ID NO: 11 and a light chain variableregion comprising a CDR2 having the amino acid sequence of SEQ ID NO:15. In other embodiments, the antibody, or antigen binding portionthereof, comprises a heavy chain variable region comprising a CDR1having the amino acid sequence of SEQ ID NO: 10 and a light chainvariable region comprising a CDR1 having the amino acid sequence ofeither SEQ ID NO: 14.

In one aspect of the invention, the present disclosure provides anisolated antibody, or antigen binding portion thereof, that binds tohuman LY6H, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR3 having theamino acid sequence of SEQ ID NO: 19 and a light chain variable regioncomprising a CDR3 having the amino acid sequence of SEQ ID NO: 21.

In some embodiments, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR2 having theamino acid sequence of SEQ ID NO: 11 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 15. Inother embodiments, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR1 having theamino acid sequence of SEQ ID NO: 18 and a light chain variable regioncomprising a CDR1 having the amino acid sequence of either SEQ ID NO:14.

In yet another aspect of the invention, the present disclosure providesan isolated antibody, or antigen binding portion thereof, that binds tohuman LY6H, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR3 having theamino acid sequence of SEQ ID NO: 25 and a light chain variable regioncomprising a CDR3 having the amino acid sequence of SEQ ID NO: 8.

In some aspects, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR2 having one ofthe amino acid sequences selected from SEQ ID NO: 24, SEQ ID NO: 65, andSEQ ID NO: 67, and a light chain variable region comprising a CDR2having the amino acid sequence of SEQ ID NO: 7. In other embodiments,the antibody, or antigen binding portion thereof, comprises a heavychain variable region comprising a CDR1 having the amino acid sequenceof SEQ ID NO: 23 and a light chain variable region comprising a CDR1having the amino acid sequence of either SEQ ID NO: 6.

In another aspect of the invention, the present disclosure provides anisolated antibody, or antigen binding portion thereof, that binds tohuman LY6H, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR3 having theamino acid sequence of SEQ ID NO: 30 and a light chain variable regioncomprising a CDR3 having the amino acid sequence of SEQ ID NO: 34.

In some aspects, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR2 having theamino acid sequence of SEQ ID NO: 29 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 33. Inother embodiments, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR1 having theamino acid sequence of SEQ ID NO: 28 and a light chain variable regioncomprising a CDR1 having the amino acid sequence of either SEQ ID NO:32.

In one aspect of the invention, the present disclosure provides anisolated antibody, or antigen binding portion thereof, that binds tohuman LY6H, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR3 having theamino acid sequence of SEQ ID NO: 36 and a light chain variable regioncomprising a CDR3 having the amino acid sequence of SEQ ID NO: 38.

In some aspects, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR2 having theamino acid sequence of SEQ ID NO: 29 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 33. Inother embodiments, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR1 having theamino acid sequence of SEQ ID NO: 28 and a light chain variable regioncomprising a CDR1 having the amino acid sequence of either SEQ ID NO:32.

In another aspect of the invention, the present disclosure provides anisolated antibody, or antigen binding portion thereof, that binds tohuman LY6H, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR3 having theamino acid sequence of SEQ ID NO: 40 and a light chain variable regioncomprising a CDR3 having the amino acid sequence of SEQ ID NO: 44.

In some aspects, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR2 having theamino acid sequence of SEQ ID NO: 29 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 43. Inother embodiments, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR1 having theamino acid sequence of SEQ ID NO: 28 and a light chain variable regioncomprising a CDR1 having the amino acid sequence of either SEQ ID NO:42.

In one aspect of the invention, the present disclosure provides anisolated antibody, or antigen binding portion thereof, that binds tohuman LY6H, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR3 having theamino acid sequence of SEQ ID NO: 48 and a light chain variable regioncomprising a CDR3 having the amino acid sequence of SEQ ID NO: 52.

In some aspects, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR2 having theamino acid sequence of SEQ ID NO: 47 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 51. Inother embodiments, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR1 having theamino acid sequence of SEQ ID NO: 46 and a light chain variable regioncomprising a CDR1 having the amino acid sequence of either SEQ ID NO:50.

In another aspect of the invention, the present disclosure provides anisolated antibody, or antigen binding portion thereof, that binds tohuman LY6H, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR3 having theamino acid sequence of SEQ ID NO: 48 and a light chain variable regioncomprising a CDR3 having the amino acid sequence of SEQ ID NO: 52.

In some aspects, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR2 having theamino acid sequence of SEQ ID NO: 29 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 51. Inother embodiments, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR1 having theamino acid sequence of SEQ ID NO: 46 and a light chain variable regioncomprising a CDR1 having the amino acid sequence of either SEQ ID NO:50.

In one aspect of the invention, the present disclosure provides anisolated antibody, or antigen binding portion thereof, that binds tohuman LY6H, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR3 having theamino acid sequence of SEQ ID NO: 55 and a light chain variable regioncomprising a CDR3 having the amino acid sequence of SEQ ID NO: 57.

In some aspects, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR2 having theamino acid sequence of SEQ ID NO: 29 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 51. Inother embodiments, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR1 having theamino acid sequence of SEQ ID NO: 28 and a light chain variable regioncomprising a CDR1 having the amino acid sequence of either SEQ ID NO:50.

In another aspect of the invention, the present disclosure provides anisolated antibody, or antigen binding portion thereof, that binds tohuman LY6H, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR3 having theamino acid sequence of SEQ ID NO: 59 and a light chain variable regioncomprising a CDR3 having the amino acid sequence of SEQ ID NO: 57.

In some aspects, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR2 having theamino acid sequence of SEQ ID NO: 29 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 51. Inother embodiments, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR1 having theamino acid sequence of SEQ ID NO: 28 and a light chain variable regioncomprising a CDR1 having the amino acid sequence of either SEQ ID NO:50.

In one aspect of the invention, the present disclosure provides anisolated antibody, or antigen binding portion thereof, that binds tohuman LY6H, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR3 having theamino acid sequence of SEQ ID NO: 63 and a light chain variable regioncomprising a CDR3 having the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR2 having one ofthe amino acid sequences selected from SEQ ID NO: 62 and SEQ ID NO: 71,and a light chain variable region comprising a CDR2 having the aminoacid sequence of SEQ ID NO: 7. In some embodiments, the antibody, orantigen binding portion thereof, comprises a heavy chain variable regioncomprising a CDR1 having the amino acid sequence of SEQ ID NO: 61 and alight chain variable region comprising a CDR1 having the amino acidsequence of either SEQ ID NO: 6.

In some aspects, the antibody, or antigen binding portion thereof, is anIgG isotype.

In some aspects, the antibody, or antigen binding portion thereof, has aK_(D) of 200 nM or less.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 4, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 3 or SEQ ID NO: 69, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 2, and a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 10, and a light chain variable regioncomprising a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 16, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:15, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:14.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 19, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 18, and a light chain variable regioncomprising a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 21, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:15, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:14.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 25, a CDR2 domain comprising one ofthe amino acid sequences selected from SEQ ID NO: 24, SEQ ID NO: 65, andSEQ ID NO: 67, and a CDR1 domain comprising the amino acid sequence ofSEQ ID NO: 23, and a light chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 8, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 7, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 6.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 30, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 29, and a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 28, and a light chain variable regioncomprising a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 34, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:33, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:32.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 36, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 29, and a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 28, and a light chain variable regioncomprising a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 38, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:33, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:32.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 40, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 29, and a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 28, and a light chain variable regioncomprising a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 44, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:43, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:42.

In one aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 48, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 47, and a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 46, and a light chain variable regioncomprising a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 52, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:51, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:50.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 48, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 29, and a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 46, and a light chain variable regioncomprising a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 52, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:51, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:50.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 55, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 29, and a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 28, and a light chain variable regioncomprising a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 57, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:51, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:50.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 59, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 29, and a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 28, and a light chain variable regioncomprising a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 57, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:51, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:50.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 63, a CDR2 domain comprising one ofthe amino acid sequence selected from SEQ ID NO: 62 and SEQ ID NO: 71,and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 61,and a light chain variable region comprising a CDR3 domain comprisingthe amino acid sequence of SEQ ID NO: 8, a CDR2 domain comprising theamino acid sequence of SEQ ID NO: 7, and a CDR1 domain comprising theamino acid sequence of SEQ ID NO: 6.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable domain comprising an amino acid sequence set forthin SEQ ID NO: 1 or SEQ ID NO: 68 and a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 5.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain comprising an amino acid sequence set forth in SEQ ID NO: 1or SEQ ID NO: 68, or a sequence having at least 90%, 95%, 96%, 97%, 98%,or 99% identity to SEQ ID NO: 1, and/or a light chain comprising anamino acid sequence set forth in SEQ ID NO: 5, or a sequence having atleast 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 9 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 13.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain comprising an amino acid sequence set forth in SEQ ID NO: 9,or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identityto SEQ ID NO: 9, and/or a light chain comprising an amino acid sequenceset forth in SEQ ID NO: 13, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 13.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 17 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 20.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain comprising an amino acid sequence set forth in SEQ ID NO:17, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 17, and/or a light chain comprising an amino acidsequence set forth in SEQ ID NO: 20, or a sequence having at least 90%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 20.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising one of the amino acid sequencesselected from SEQ ID NO: 22, SEQ ID NO: 64, and SEQ ID NO: 66, and alight chain variable region comprising the amino acid sequence of SEQ IDNO: 26.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain comprising an amino acid sequence set forth in one of SEQ IDNO: 22, SEQ ID NO: 64, and SEQ ID NO: 66, or a sequence having at least90%, 95%, 96%, 97%, 98%, or 99% identity to one of SEQ ID NO: 22, SEQ IDNO: 64, and SEQ ID NO: 66, and/or a light chain comprising an amino acidsequence set forth in SEQ ID NO: 29, or a sequence having at least 90%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 27 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 31.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain comprising an amino acid sequence set forth in SEQ ID NO:27, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 27, and/or a light chain comprising an amino acidsequence set forth in SEQ ID NO: 31, or a sequence having at least 90%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 31.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 35 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 37.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain comprising an amino acid sequence set forth in SEQ ID NO:35, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 35, and/or a light chain comprising an amino acidsequence set forth in SEQ ID NO: 37, or a sequence having at least 90%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 37.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 39 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 41.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain comprising an amino acid sequence set forth in SEQ ID NO:39, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 39, and/or a light chain comprising an amino acidsequence set forth in SEQ ID NO: 41, or a sequence having at least 90%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 41.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 45 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 49.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain comprising an amino acid sequence set forth in SEQ ID NO:45, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 45, and/or a light chain comprising an amino acidsequence set forth in SEQ ID NO: 49, or a sequence having at least 90%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 49.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 53 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 49.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain comprising an amino acid sequence set forth in SEQ ID NO:53, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 53, and/or a light chain comprising an amino acidsequence set forth in SEQ ID NO: 49, or a sequence having at least 90%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 49.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 54 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 56.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain comprising an amino acid sequence set forth in SEQ ID NO:54, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 54, and/or a light chain comprising an amino acidsequence set forth in SEQ ID NO: 56, or a sequence having at least 90%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 56.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 58 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 56.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain comprising an amino acid sequence set forth in SEQ ID NO:58, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 58, and/or a light chain comprising an amino acidsequence set forth in SEQ ID NO: 56, or a sequence having at least 90%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 56.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 60 or SEQ ID NO:70 and a light chain variable region comprising theamino acid sequence of SEQ ID NO: 26.

In another aspect of the invention, the present disclosure provides ananti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain comprising an amino acid sequence set forth in SEQ ID NO: 60or SEQ ID NO:70, or a sequence having at least 90%, 95%, 96%, 97%, 98%,or 99% identity to SEQ ID NO: 60 or SEQ ID NO:70, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 26, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 26.

In another aspect of the invention, the present disclosure provides anantibody, or antigen-binding portion thereof, that binds to the sameepitope as the antibody, or antigen-binding portion thereof, of any oneof the preceding claims.

In another aspect of the invention, the present disclosure provides anisolated nucleic acid encoding an antibody, or antigen binding portionthereof, of any one of the preceding claims.

In one aspect of the invention, the present disclosure provides apharmaceutical composition comprising the antibody, or antigen bindingportion thereof, of any one of the preceding claims, and apharmaceutically acceptable carrier.

In another aspect of the invention, the present disclosure provides anantibody, or antigen binding portion thereof, of any one of thepreceding claims, conjugated to at least one drug.

In some aspects, the at least one drug is selected from the groupconsisting of an anti-apoptotic agent, a mitotic inhibitor, a DNAdamaging agent, an anti-tumor antibiotic, an immunomodulating agent, anucleic acid for gene therapy, an anti-angiogenic agent, anantimetabolite, a boron-containing agent, a chemoprotective agent, ahormone agent, an antihormone agent, a corticosteroid, a photoactivetherapeutic agent, an oligonucleotide, a radionuclide agent, aradiosensitizer, a topoisomerase inhibitor, and a tyrosine kinaseinhibitor. In other embodiments, the at least one drug is conjugated tothe antibody, or antigen-binding portion thereof, via a linker. Inanother embodiment, the linker is a cleavable linker. In yet otherembodiments, the linker is a non-cleavable linker.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising a CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, a CDR2 domain comprising one of the amino acid sequencesselected from SEQ ID NO: 3 and SEQ ID NO: 69, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 2, and a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising a CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 63, a CDR2 domain comprising one of the amino acid sequencesselected from SEQ ID NO: 62 and SEQ ID NO:71, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 61, and a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising a CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQID NO: 10, and a light chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 16, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 15, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 14.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising a CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 19, a CDR2 domain comprising the amino acid sequence of SEQID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQID NO: 18, and a light chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 21, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 15, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 14.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising a CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 25, a CDR2 domain comprising one of the amino acid sequencesselected from SEQ ID NO: 24, SEQ ID NO: 65 and SEQ ID NO: 67, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 23, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising a CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 30, a CDR2 domain comprising the amino acid sequence of SEQID NO: 29, and a CDR1 domain comprising the amino acid sequence of SEQID NO: 28, and a light chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 34, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 33, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 32.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising a CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 36, a CDR2 domain comprising the amino acid sequence of SEQID NO: 29, and a CDR1 domain comprising the amino acid sequence of SEQID NO: 28, and a light chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 38, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 33, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 32.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising a CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 40, a CDR2 domain comprising the amino acid sequence of SEQID NO: 29, and a CDR1 domain comprising the amino acid sequence of SEQID NO: 28, and a light chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 44, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO:43, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 42.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising a CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 48, a CDR2 domain comprising the amino acid sequence of SEQID NO: 47, and a CDR1 domain comprising the amino acid sequence of SEQID NO: 46, and a light chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 52, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 51, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 50.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising a CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 48, a CDR2 domain comprising the amino acid sequence of SEQID NO: 29, and a CDR1 domain comprising the amino acid sequence of SEQID NO: 46, and a light chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 52, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 51, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 50.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising a CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 55, a CDR2 domain comprising the amino acid sequence of SEQID NO: 29, and a CDR1 domain comprising the amino acid sequence of SEQID NO: 28, and a light chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 57, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 51, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 50.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising a CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 59, a CDR2 domain comprising the amino acid sequence of SEQID NO: 29, and a CDR1 domain comprising the amino acid sequence of SEQID NO: 28, and a light chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 57, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 51, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 50.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variabledomain comprising an amino acid sequence set forth in SEQ ID NO: 1 orSEQ ID NO: 68, and a light chain variable domain comprising an aminoacid sequence set forth in SEQ ID NO: 5.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain comprisingan amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 68, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 1 or SEQ ID NO: 68, and/or a light chain comprising an amino acidsequence set forth in SEQ ID NO: 5, or a sequence having at least 90%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 9 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:13.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain comprisingan amino acid sequence set forth in SEQ ID NO: 9, or a sequence havingat least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9,and/or a light chain comprising an amino acid sequence set forth in SEQID NO: 13, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 13.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 60 or SEQ ID NO:70 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 26.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain comprisingan amino acid sequence set forth in SEQ ID NO: 60 or SEQ ID NO: 70, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 60 or SEQ ID NO: 70, and/or a light chain comprising an aminoacid sequence set forth in SEQ ID NO: 26, or a sequence having at least90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 17 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:20.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain comprisingan amino acid sequence set forth in SEQ ID NO: 17, or a sequence havingat least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 17,and/or a light chain comprising an amino acid sequence set forth in SEQID NO: 20, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 20.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising one of the amino acid sequences selected from SEQ IDNO: 22, SEQ ID NO: 64, and SEQ ID NO: 66 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 26.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain comprisingan amino acid sequence set forth in one of SEQ ID NO: 22, SEQ ID NO: 64and SEQ ID NO: 66, or a sequence having at least 90%, 95%, 96%, 97%,98%, or 99% identity to one of SEQ ID NO: 22, SEQ ID NO: 64 and SEQ IDNO: 66, and/or a light chain comprising an amino acid sequence set forthin SEQ ID NO: 26, or a sequence having at least 90%, 95%, 96%, 97%, 98%,or 99% identity to SEQ ID NO: 26.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 27 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:31.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain comprisingan amino acid sequence set forth in SEQ ID NO: 27, or a sequence havingat least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 27,and/or a light chain comprising an amino acid sequence set forth in SEQID NO: 31, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 31.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 35 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:37.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain comprisingan amino acid sequence set forth in SEQ ID NO: 35, or a sequence havingat least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 35,and/or a light chain comprising an amino acid sequence set forth in SEQID NO: 37, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 37.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 39 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:41.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain comprisingan amino acid sequence set forth in SEQ ID NO: 39, or a sequence havingat least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 39,and/or a light chain comprising an amino acid sequence set forth in SEQID NO: 41, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 41.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 45 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:49.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain comprisingan amino acid sequence set forth in SEQ ID NO: 45, or a sequence havingat least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 45,and/or a light chain comprising an amino acid sequence set forth in SEQID NO: 49, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 49.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 53 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:49.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain comprisingan amino acid sequence set forth in SEQ ID NO: 53, or a sequence havingat least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 53,and/or a light chain comprising an amino acid sequence set forth in SEQID NO: 49, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 49.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 54 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:56.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain comprisingan amino acid sequence set forth in SEQ ID NO: 54, or a sequence havingat least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 54,and/or a light chain comprising an amino acid sequence set forth in SEQID NO: 56, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 56.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 58 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:56.

In another aspect of the invention, the present disclosure provides anantibody drug conjugate (ADC) comprising an antibody, or antigen bindingportion thereof, conjugated to at least one drug, wherein the antibody,or antigen binding portion thereof, comprises a heavy chain comprisingan amino acid sequence set forth in SEQ ID NO: 58, or a sequence havingat least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 58,and/or a light chain comprising an amino acid sequence set forth in SEQID NO: 56, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 56.

In some embodiments, the at least one drug is conjugated via a linker.In other embodiments, the linker is a cleavable linker. In yet otherembodiments, the linker is a non-cleavable linker.

In some embodiments, the antibody, or antigen binding portion thereof,is an IgG1 isotype. In another aspect of the invention, the presentdisclosure provides a pharmaceutical composition comprising an ADCmixture comprising a plurality of the ADC as described herein, and apharmaceutically acceptable carrier.

In some embodiments, the ADC mixture has an average drug to antibodyratio (DAR) of 0 to 8.

In another aspect of the invention, the present disclosure provides amethod of treating cancer using the antibody, or antigen binding portionthereof, as described herein, or the antibody drug conjugate, asdescribed herein.

In some embodiments, the cancer is small cell lung cancer. In oneembodiment, the small cell lung cancer is classic small cell lungcancer. In another embodiment, the small cell lung cancer is a variantsmall cell lung cancer. In one embodiment, classic SCLC cell linesinclude but are not limited to, for example, the following cell lines:NCI-H60, NCI-H64, NCI-H69, NCI-H209, DMS79, and NCI-H220. In anotherembodiment, variant SCLC cell lines include but are not limited to, forexample, the following cell lines: NCI-H446, NCI-H526, NCI-H524, andNCI-H82,

In some embodiments, the cancer is selected from the groups consistingof gastrointestinal stromal tumor (GIST), T cell acute lymphoblasticleukemia (T-ALL), glioblastoma, and soft tissue sarcoma. In yet otherembodiments, the cancer is selected from the group consisting of breastcancer, brain and CNS cancer, head and neck cancer, bladder cancer,renal cancer, ovarian cancer, gastric cancer, esophageal cancer,prostate cancer, uterine cancer, endometrial cancer, pancreatic cancer,liver cancer, colorectal cancer, cervical cancer, or non-small cell lungcancer (NSCLC).

In another aspect of the invention, the present disclosure provides amethod of inhibiting or decreasing tumor growth using the antibody, orantigen binding portion thereof, as described herein, or the antibodydrug conjugate, as described herein.

In some embodiments, the tumor is small cell lung cancer tumor. In otherembodiments, the tumor is gastrointestinal stromal tumor (GIST),glioblastoma, or soft tissue sarcoma.

In some embodiments, the present disclosure provides a method forinhibiting or decreasing the progression of a cancer in a subject thatdoes not generally form a solid tumor, said method comprisingadministering an effective amount of the antibody or antigen bindingportion thereof, as described herein, or the ADC, as described herein,to the subject having the cancer, such that the progression of thecancer is inhibited or decreased. In a particular embodiment, the canceris T cell acute lymphoblastic leukemia (T-ALL).

In some embodiments, the antibody or antigen binding portion thereof orthe ADC is administered in combination with an additional agent or anadditional therapy. In other embodiments, the additional agent is animmune checkpoint inhibitor. In yet another embodiment, the immunecheckpoint inhibitor is an antibody. In another embodiment, the antibodyis selected from the group consisting of an anti-PD1 antibody, ananti-PD-L1 antibody or an anti-CTLA-4 antibody. In other embodiments,the additional therapy is radiation. In yet another embodiment, theadditional agent is a chemotherapeutic agent. In some embodiments, thecancer or tumor is characterized as having LY6H expression oroverexpression.

In some embodiments, the antibodies, or antigen binding portionsthereof, of the invention, bind to LY6H with a K_(d) of about 2,000 nMor less, about 1,000 nM or less, about 500 nM or less, about 200 nM orless, about 100 nM or less, about 75 nM or less, about 25 nM or less,about 21 nM or less, about 12 nM or less, about 11 nM or less, about 10nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less,about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nMor less, about 2 nM or less, about 1 nM or less, about 0.5 nM or less,about 0.3 nM or less, about 0.1 nM or less, or about 0.01 nM or less, orabout 0.001 nM or less.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that LY6H is differentially expressed in primary SCLCtumors. iBAQ (intensity-based absolute quantification) data ispresented. Triangles represent SCLC tumors, squares represent SCLCnormal adjacent tissue (NAT), and circles represent normal lung tissue.

FIG. 2 shows that anti-LY6H antibody (12G7) binds both LY6H positiveSCLC and T cell acute lymphoblastic leukemia (T-ALL) cell lines. Dottedlines represent LY6H antibody.

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, and FIG. 3E show internalization andin vitro efficacy of anti-LY6H antibodies. Anti-LY6H antibodies wereevaluated for cell surface binding to 293 cells expressing human (A),Macaca fascicularis (B), rat (C), mouse LY6H (D), and mock transfection(E) by flow. ab55472 (abeam) and 22H11 are mouse antibodies againsthuman LY6H. Dotted lines represent LY6H antibody. hIG1 (antibody againstHBV surface antigen) was included as a negative control (solid lines).FIG. 3A shows binding of anti-LY6H antibodies to human LY6H. FIG. 3Bshows binding of anti-LY6H antibodies to Macaca fascicularis LY6H. FIG.3C shows binding of anti-LY6H antibodies to rat LY6H. FIG. 3D showsbinding of anti-LY6H antibodies to mouse LY6H. FIG. 3E shows binding ofanti-LY6H antibodies to parental 293.

FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D show half maximal effectiveconcentration (EC₅₀) of anti-LY6H antibodies in SCLC and T-ALL celllines.

FIG. 5 shows on cell antibody affinity of anti-LY6H antibodies. 1 nM PEconjugated 12G7 and 1F8 antibodies were incubated with 2 fold serialdilutions (from 100 nM to 0.05 nM) of unconjugated Ab in binding buffer.Serial diluted antibodies were incubated with 293 cells expressingeither human or Macaca fascicularis on ice for 4 hours, after a briefwash. Acquisition of the MFI (Median Fluorescent Intensity) wasperformed on a MACSQuant® Flow Cytometers (Miltenyi Biotec). On cellaffinity (Kd) was calculated using dose-response EC50 shift by globalfitting from GraphPad Prism™.

FIG. 6 shows internalization of 12G7 into H446 cells. Live H446 cellswere incubated with 12G7 for 2 hours, cells were then fixed,permeablized, and co-stained with LAMP1 antibody.

FIG. 7A, FIG. 7B, and FIG. 7C show internalization of anti-LY6Hantibodies and in vitro efficacy.

FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, FIG. 8F, and FIG. 8G showin vitro efficacy of anti-LY6H ADC in SCLC and T-ALL cell lines.3000-5000 cells were seeded in 96 well plate, and treated with MMAE orPBD conjugated antibodies for 5 days. Then cells were lysed by CellTiterGlo 2.0™ (Promega), results were recorded by luminometer. CBIgG1(Anti-Hen Egg Lysozyme antibody, CrownBio) and hIgG1 (anti-HBV surfaceAg antibody) conjugated antibodies were included as negative controls.IC50 values are listed at the bottom of each graph.

FIG. 9A, FIG. 9B, and FIG. 9C show MMAE conjugated antibody inhibitstumor growth in vivo. FIG. 9A depicts efficacy of MMAE conjugatedantibodies in H446 xenograft model. FIG. 9B depicts Kaplan-Meiersurvival curve of the study. FIG. 9C shows the body weight of the micein the study.

FIG. 10A, FIG. 10B, and FIG. 10C show Tesirine PBD conjugated antibodyinhibits tumor growth in vivo. FIG. 10A shows efficacy of PBD conjugatedantibodies in H446 xenograft model. FIG. 10B shows a Kaplan-Meiersurvival curve of the study. FIG. 10C shows the mice body weight of thestudy.

FIG. 11A, FIG. 11B, and FIG. 11C show that MMAE conjugated antibodyshowed limited tumor growth inhibition in vivo. FIG. 11A shows efficacyof MMAE conjugated antibodies in H526 xenograft model. FIG. 11B shows aKaplan-Meier survival curve of the study. FIG. 11C shows the body weightof the mice in the study.

FIG. 12A, FIG. 12B, and FIG. 12C show Tesirine PBD conjugated antibodyinhibits tumor growth in vivo. FIG. 12A shows efficacy of PBD conjugatedantibodies in H526 xenograft model. FIG. 12B shows a Kaplan-Meiersurvival curve of the study. FIG. 12C shows the body weight of the micein the study.

FIG. 13A, FIG. 13B, and FIG. 13C show a single dose of Tesirine PBDconjugated antibody inhibits tumor growth in vivo. FIG. 13A showsefficacy of PBD conjugated antibodies in DMS79 xenograft model. FIG. 13Bshows a Kaplan-Meier survival curve of the study. FIG. 13C shows thebody weight of the mice in the study.

FIG. 14A, FIG. 14B, and FIG. 14C show in vivo efficacy of a single doseof Tesirine PBD conjugated antibody in H446 cells. FIG. 14A showsefficacy of PBD conjugated antibodies in H446 xenograft model. FIG. 14Bshows a Kaplan-Meier survival curve of the study. FIG. 14C shows thebody weight of the mice in the study.

FIG. 15A, FIG. 15B, and FIG. 15C show isobologram analysis ofcombination treatments of anti-LY6H-PBD with olaparib, cisplatin, oretoposide in DMS79 cells. Combination treatments were performed with aconstant concentration of 12G7-PBD and titrating doses of olaparib (A),cisplatin (B) or etoposide (C). IC50 values were recorded for singletreatments or combinations to calculate isobologram points. FIG. 15Ashows PBD in combination with olaparib. FIG. 15B shows PBD incombination with cisplatin. FIG. 15C shows PBD in combination withetoposide.

FIG. 16A, FIG. 16B, and FIG. 16C show isobologram analysis ofcombination treatments of anti-LY6H-PBD with olaparib, cisplatin, oretoposide in CORL95 cells. Combination treatments were performed with aconstant concentration of 12G7-PBD and titrating doses of olaparib (A),cisplatin (B) or etoposide (C). IC50 values were recorded for singletreatments or combinations to calculate isobologram points. FIG. 16Ashows PBD in combination with olaparib. FIG. 16B shows PBD incombination with cisplatin. FIG. 16C shows PBD in combination withetoposide.

FIG. 17A, FIG. 17B, and FIG. 17C show isobologram analysis ofcombination treatments of anti-LY6H-PBD with olaparib, cisplatin, oretoposide in NCI-H1092 cells. Combination treatments were performed witha constant concentration of 12G7-PBD and titrating doses of olaparib(A), cisplatin (B) or etoposide (C). IC50 values were recorded forsingle treatments or combinations to calculate isobologram points. FIG.17A shows PBD in combination with olaparib. FIG. 17B shows PBD incombination with cisplatin. FIG. 17C shows PBD in combination withetoposide.

DETAILED DESCRIPTION

Various aspects of the disclosure relate to anti-LY6H antibodies andantibody fragments, anti-LY6H ADCs, and pharmaceutical compositionsthereof, as well as nucleic acids, recombinant expression vectors andhost cells for making such antibodies and fragments. Methods of usingthe antibodies and ADCs described herein to detect LY6H, in particularhuman LY6H, to bind to and inhibit LY6H on LY6H expressing cells,including cancer cells, to undergo internalization in and inhibit LY6Hexpressing cells, e.g., LY6H expressing tumor and cancer cells, tomodulate an immune response in vivo, and/or to treat LY6H-associateddisorders, e.g., cancer, including, but not limited to, small cell lungcancer (SCLC). In one embodiment, the anti-LY6H antibodies or ADCs ofthe invention are administered in combination with one or more immunecheckpoint inhibitors (e.g., antibody or small molecule immunecheckpoint inhibitors) for the treatment of a cancer. In anotherembodiment of the invention, anti-LY6H antibody drug conjugates (ADCs)of the invention (e.g., the LY6H antibodies of the invention conjugatedto a toxin) are internalized and induce cell death of cells endogenouslyexpressing LY6H.

I. Definitions

In order that the invention may be more readily understood, certainterms are first defined. In addition, it should be noted that whenever avalue or range of values of a parameter are recited, it is intended thatvalues and ranges intermediate to the recited values are also intendedto be part of this invention.

The terms “lymphocyte antigen 6 complex, locus H antibody,” “LymphocyteAntigen 6H antibody,” or “anti-LY6H antibody”, used interchangeablyherein, refer to an antibody that specifically binds to LY6H, e.g.,human LY6H. An antibody “which binds” an antigen of interest, i.e.,LY6H, is one capable of binding that antigen with sufficient affinitysuch that the antibody is useful in targeting a cell expressing theantigen. In a preferred embodiment, the antibody specifically binds tohuman LY6H (hLY6H). Examples of anti-LY6H antibodies are disclosed inthe Examples, below. Unless otherwise indicated, the term “anti-LY6Hantibody” is meant to refer to an antibody which binds to wild typeLY6H, a variant, or an isoform of LY6H.

Several different isoforms of LY6H have been identified. An exemplaryamino acid sequence of wild type human LY6H, which contains 140 aminoacids, is provided below as SEQ ID NO: 123 (GenBank LY6H_HUMAN).

        10          20        30         40MLPAAMKGLG LALLAVLLCS APAHGLWCQD CTLTTNSSHC        50          60        70         80TPKQCQPSDT VCASVRITDP SSSRKDHSVN KMCASSCDFV        90        100        110        120KRHFFSDYLM GFINSGILKV DVDCCEKDLC NGAAGAGHSP        130        140WALAGGLLLS LGPALLWAGP

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of a LY6H antibody or an ADC with asecond chemical species, mean that the interaction is dependent upon thepresence of a particular structure (e.g., an antigenic determinant orepitope) on the chemical species; for example, an antibody recognizesand binds to a specific protein structure rather than to proteinsgenerally. If an antibody or ADC is specific for epitope “A”, thepresence of a molecule containing epitope A (or free, unlabeled A), in areaction containing labeled “A” and the antibody, will reduce the amountof labeled A bound to the antibody or ADC.

In one embodiment, the phrase “specifically binds to LY6H” or “specificbinding to LY6H”, as used herein, refers to the ability of an anti-LY6Hantibody or ADC to interact with LY6H (human, monkey, rat or mouse LY6H)with a dissociation constant (K_(D)) of about 2,000 nM or less, about1,000 nM or less, about 500 nM or less, about 200 nM or less, about 100nM or less, about 75 nM or less, about 25 nM or less, about 21 nM orless, about 12 nM or less, about 11 nM or less, about 10 nM or less,about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nMor less, about 5 nM or less, about 4 nM or less, about 3 nM or less,about 2 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.3nM or less, about 0.1 nM or less, about 0.01 nM or less, or about 0.001nM or less. In another embodiment, the phrase “specifically binds toLY6H” or “specific binding to LY6H”, as used herein, refers to theability of an anti-LY6H antibody or ADC to interact with LY6H (human,monkey, rat or mouse LY6H) with a dissociation constant (K_(D)) ofbetween about 1 pM (0.001 nM) to 2,000 nM, between about 500 pM (0.5 nM)to 1,000 nM, between about 500 pM (0.5 nM) to 500 nM, between about 1nM) to 200 nM, between about 1 nM to 100 nM, between about 1 nM to 50nM, between about 1 nM to 20 nM, or between about 1 nM to 5 nM. In oneembodiment, K_(D) is determined by surface plasmon resonance. In oneembodiment, K_(D) is determined by surface plasmon resonance orBio-Layer Interferometry, or by any other method known in the art.Bio-Layer Interferometry refers to an optical phenomenon that allows forthe analysis of real-time biospecific interactions by measuring theinterference patterns of reflected white light, for example using theOctet™ system (ForteBio, Pall Corp. Fremont, Calif.). For furtherdescription of the Octet™ system, see Li, B et al. (2011) J. Pharm.Biomed. Anal. 54(2):286-294 and Abdiche, Y. N., et al. (2009) Anal.Biochem. 386(2):172-180, the contents of which are incorporated hereinby reference.

The term “antibody” broadly refers to an immunoglobulin (Ig) molecule,generally comprised of four polypeptide chains, two heavy (H) chains andtwo light (L) chains, or any functional fragment, mutant, variant, orderivative thereof, that retains the essential target binding featuresof an Ig molecule. Such mutant, variant, or derivative antibody formatsare known in the art. Non-limiting embodiments of which are discussedbelow.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY) and class (e.g., IgG1, IgG2, IgG 3, IgG4, IgA1and IgA2) or subclass.

The term “antigen binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., hLY6H). It has been shown that the antigen binding function of anantibody can be performed by fragments of a full-length antibody. Suchantibody embodiments may also be bispecific, dual specific, ormulti-specific formats; specifically binding to two or more differentantigens. Examples of binding fragments encompassed within the term“antigen binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publicationWO 90/05144 A1 herein incorporated by reference), which comprises asingle variable domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen binding portion” ofan antibody. In certain embodiments, scFv molecules may be incorporatedinto a fusion protein. Other forms of single chain antibodies, such asdiaboclies are also encompassed. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc.Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994)Structure 2:1121-1123). Such antibody binding portions are known in theart (Kontermann and Dubel eds., Antibody Engineering (2001)Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

The term “antibody construct” as used herein refers to a polypeptidecomprising one or more the antigen binding portions disclosed hereinlinked to a linker polypeptide or an immunoglobulin constant domain.Linker polypeptides comprise two or more amino acid residues joined bypeptide bonds and are used to link one or more antigen binding portions.Such linker polypeptides are well known in the art (see e.g., Holliger,P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). An immunoglobulin constantdomain refers to a heavy or light chain constant domain. Antibodyportions, such as Fab and F(ab′)₂ fragments, can be prepared from wholeantibodies using conventional techniques, such as papain or pepsindigestion, respectively, of whole antibodies. Moreover, antibodies,antibody portions and immunoadhesion molecules can be obtained usingstandard recombinant DNA techniques, as described herein.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds LY6H is substantially free of antibodies that specifically bindantigens other than LY6H). An isolated antibody that specifically bindsLY6H may, however, have cross-reactivity to other antigens, such as LY6Hmolecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a nonhuman species (e.g.,a mouse) but in which at least a portion of the VH and/or VL sequencehas been altered to be more “human-like”, i.e., more similar to humangermline variable sequences. In particular, the term “humanizedantibody” is an antibody or a variant, derivative, analog or fragmentthereof which immunospecifically binds to an antigen of interest andwhich comprises a framework (FR) region having substantially the aminoacid sequence of a human antibody and a complementary determining region(CDR) having substantially the amino acid sequence of a non-humanantibody. As used herein, the term “substantially” in the context of aCDR refers to a CDR having an amino acid sequence at least 80%,preferably at least 85%, at least 90%, at least 95%, at least 98% or atleast 99% identical to the amino acid sequence of a non-human antibodyCDR. A humanized antibody comprises substantially all of at least one,and typically two, variable domains (Fab, Fab′, F(ab′)₂, FabC, Fv) inwhich all or substantially all of the CDR regions correspond to those ofa non-human immunoglobulin (i.e., donor antibody) and all orsubstantially all of the framework regions are those of a humanimmunoglobulin consensus sequence. Preferably, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. In some embodiments, ahumanized antibody contains both the light chain as well as at least thevariable domain of a heavy chain. The antibody also may include the CHLhinge, CH2, CH3, and CH4 regions of the heavy chain. In someembodiments, a humanized antibody only contains a humanized light chain.In other embodiments, a humanized antibody only contains a humanizedheavy chain. In specific embodiments, a humanized antibody only containsa humanized variable domain of a light chain and/or humanized heavychain.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including without limitation IgG1, IgG2, IgG3 and IgG4. The humanizedantibody may comprise sequences from more than one class or isotype, andparticular constant domains may be selected to optimize desired effectorfunctions using techniques well-known in the art.

The terms “Kabat numbering,” “Kabat definitions,” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e., hypervariable) than other amino acid residues inthe heavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain (HC) and the light chain(LC), which are designated CDR1, CDR2 and CDR3 (or specifically HC CDR1,HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3), for each of thevariable regions. The term “CDR set” as used herein refers to a group ofthree CDRs that occur in a single variable region capable of binding theantigen. The exact boundaries of these CDRs have been defineddifferently according to different systems. The system described byKabat (Kabat et al., Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987) and (1991)) notonly provides an unambiguous residue numbering system applicable to anyvariable region of an antibody, but also provides precise residueboundaries defining the three CDRs. These CDRs may be referred to asKabat CDRs. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol.196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) foundthat certain sub-portions within Kabat CDRs adopt nearly identicalpeptide backbone conformations, despite having great diversity at thelevel of amino acid sequence. These sub-portions were designated as L1,L2 and L3 or H1, H2 and H3 where the “L” and the “H” designates thelight chain and the heavy chains regions, respectively. These regionsmay be referred to as Chothia CDRs, which have boundaries that overlapwith Kabat CDRs.

Other boundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):732-45 (1996)). Still other CDR boundary definitions may notstrictly follow one of the above systems, but will nonetheless overlapwith the Kabat CDRs, although they may be shortened or lengthened inlight of prediction or experimental findings that particular residues orgroups of residues or even entire CDRs do not significantly impactantigen binding. The methods used herein may utilize CDRs definedaccording to any of these systems, although preferred embodiments useKabat or Chothia defined CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, CDR-L2,and CDR-L3 of light chain and CDR-H1, CDR-H2, and CDR-H3 of heavy chain)also divide the framework regions on the light chain and the heavy chaininto four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in whichCDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, andCDR3 between FR3 and FR4. Without specifying the particular sub-regionsas FR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In a preferred embodiment,such mutations, however, will not be extensive. Usually, at least 80%,preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (See e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family ofimmunoglobulins, each position in the consensus sequence is occupied bythe amino acid occurring most frequently at that position in the family.If two amino acids occur equally frequently, either can be included inthe consensus sequence.

“Percent (%) amino acid sequence identity” with respect to a peptide orpolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the specific peptide or polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. In one embodiment, the disclosure includes anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to an amino acid sequence described herein.

The term “multivalent antibody” is used herein to denote an antibodycomprising two or more antigen binding sites. In certain embodiments,the multivalent antibody may be engineered to have the three or moreantigen binding sites, and is generally not a naturally occurringantibody.

The term “multispecific antibody” refers to an antibody capable ofbinding two or more unrelated antigens.

The term “dual variable domain” or “DVD,” as used interchangeablyherein, are antigen binding proteins that comprise two or more antigenbinding sites and are tetravalent or multivalent binding proteins. SuchDVDs may be monospecific, i.e., capable of binding one antigen ormultispecific, i.e. capable of binding two or more antigens. DVD bindingproteins comprising two heavy chain DVD polypeptides and two light chainDVD polypeptides are referred to a DVD Ig. Each half of a DVD Igcomprises a heavy chain DVD polypeptide, and a light chain DVDpolypeptide, and two antigen binding sites. Each binding site comprisesa heavy chain variable domain and a light chain variable domain with atotal of 6 CDRs involved in antigen binding per antigen binding site. Inone embodiment, the CDRs described herein are used in an anti-LY6H DVD.

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody or ADC for an antigen, for example,an anti-LY6H antibody that binds to a LY6H antigen. In one embodiment,an anti-LY6H antibody or anti-LY6H ADC activity includes, but is notlimited to, binding to LY6H in vitro; binding to LY6H on cellsexpressing LY6H in vivo (such as, for example, LY6H expressing tumor andcancer cells); modulating immune response in vivo; undergoinginternalization in and inhibiting LY6H expressing cells, e.g., LY6Hexpressing tumor and cancer cells; inducing cell death in cellsexpressing LY6H, including LY6H expressing tumor and cancer cells;inhibiting cancer cell invasion and metastasis; decreasing or inhibitingcancer, e.g., small cell lung cancer (SCLC), gastrointestinal stromaltumor (GIST), T cell acute lymphoblastic leukemia (T-ALL), glioblastoma,and soft tissue sarcoma; and decreasing or inhibiting tumor cellularproliferation or tumor growth in vivo. In some embodiments, the tumorcan be a LY6H negative tumor or a LY6H positive tumor. In oneembodiment, an anti-LY6H antibody is capable of being internalized intoa cell expressing LY6H.

In one embodiment, the antibody, or antigen-binding portion thereof, iscapable of inducing antibody dependent cellular cytotoxicity (ADCC). Inone embodiment, the antibody, or antigen-binding portion thereof, is notcapable of inducing ADCC.

In one embodiment, the antibody, or antigen-binding portion thereof, iscapable of inducing complement-dependent cytotoxicity (CDC). In oneembodiment, the antibody, or antigen-binding portion thereof, is notcapable of inducing CDC.

In one embodiment, the antibody, or antigen-binding portion thereof, iscapable of inducing ADCC and CDC. In one embodiment, the antibody, orantigen-binding portion thereof, is not capable of inducing ADCC or CDC.

The term “epitope” refers to a region of an antigen that is bound by anantibody, antibody fragment, or ADC. In certain embodiments, epitopedeterminants include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and/or specific charge characteristics. In certainembodiments, an antibody is said to specifically bind an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin.51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson,B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al.(1991) Anal. Biochem. 198:268-277.

The term “k_(on)” or “k_(a)”, as used herein, is intended to refer tothe on rate constant for association of an antibody to the antigen toform the antibody/antigen complex.

The term “k_(off)” or “k_(d)”, as used herein, is intended to refer tothe off rate constant for dissociation of an antibody from theantibody/antigen complex.

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction. K_(D) is calculated by k_(a)/k_(d). In one embodiment, theantibodies of the invention have a K_(D) of about 2,000 nM or less,about 1,000 nM or less, about 500 nM or less, about 200 nM or less,about 100 nM or less, about 75 nM or less, about 25 nM or less, about 21nM or less, about 12 nM or less, about 11 nM or less, about 10 nM orless, about 9 nM or less, about 8 nM or less, about 7 nM or less, about6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM orless, about 2 nM or less, about 1 nM or less, about 0.5 nM or less,about 0.3 nM or less, about 0.1 nM or less, about 0.01 nM or less, orabout 0.001 nM or less.

The term “competitive binding”, as used herein, refers to a situation inwhich a first antibody competes with a second antibody, for a bindingsite on a third molecule, e.g., an antigen. In one embodiment,competitive binding between two antibodies is determined using FACSanalysis.

The term “competitive binding assay” is an assay used to determinewhether two or more antibodies bind to the same epitope. In oneembodiment, a competitive binding assay is a competition fluorescentactivated cell sorting (FACS) assay which is used to determine whethertwo or more antibodies bind to the same epitope by determining whetherthe fluorescent signal of a labeled antibody is reduced due to theintroduction of a non-labeled antibody, where competition for the sameepitope will lower the level of fluorescence.

The term “labeled antibody” as used herein, refers to an antibody, or anantigen binding portion thereof, with a label incorporated that providesfor the identification of the binding protein, e.g., an antibody.Preferably, the label is a detectable marker, e.g., incorporation of aradiolabeled amino acid or attachment to a polypeptide of biotinylmoieties that can be detected by marked avidin (e.g., streptavidincontaining a fluorescent marker or enzymatic activity that can bedetected by optical or colorimetric methods). Examples of labels forpolypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ³⁵, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm); fluorescent labels (e.g., FITC,rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradishperoxidase, luciferase, alkaline phosphatase); chemiluminescent markers;biotinyl groups; predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags); andmagnetic agents, such as gadolinium chelates.

The term “antibody-drug-conjugate” or “ADC” refers to a binding protein,such as an antibody or antigen binding fragment thereof, chemicallylinked to one or more chemical drug(s) (also referred to herein asagent(s)) that may optionally be therapeutic or cytotoxic agents. In apreferred embodiment, an ADC includes an antibody, a cytotoxic ortherapeutic drug, and a linker that enables attachment or conjugation ofthe drug to the antibody. An ADC typically has anywhere from 1 to 8drugs conjugated to the antibody, including drug loaded species of 2, 4,6, or 8. Non-limiting examples of drugs that may be included in the ADCsare mitotic inhibitors, antitumor antibiotics, immunomodulating agents,vectors for gene therapy, alkylating agents, antiangiogenic agents,antimetabolites, boron-containing agents, chemoprotective agents,hormones, antihormone agents, corticosteroids, photoactive therapeuticagents, oligonucleotides, radionuclide agents, topoisomerase inhibitors,tyrosine kinase inhibitors, and radiosensitizers.

The terms “lymphocyte antigen 6 complex, locus H antibody drugconjugate,” “Lymphocyte Antigen 6H antibody drug conjugate,” “anti-LY6Hantibody drug conjugate,” or “anti-LY6H ADC”, used interchangeablyherein, refer to an ADC comprising an antibody that specifically bindsto LY6H, whereby the antibody is conjugated to one or more chemicalagent(s) or payloads. In one embodiment, the chemical agent is linked tothe antibody via a linker.

The term “drug-to-antibody ratio” or “DAR” refers to the number ofdrugs, e.g., IGN, auristatin, or maytansinoid, attached to the antibodyof the ADC. The DAR of an ADC can range from 1 to 8, although higherloads, e.g., 10, are also possible depending on the number of linkagesite on an antibody. The term DAR may be used in reference to the numberof drugs loaded onto an individual antibody, or, alternatively, may beused in reference to the average or mean DAR of a group of ADCs.

The term “LY6H associated disorder,” as used herein, includes anydisorder or disease (including proliferative disorders, e.g., cancer)that is marked, diagnosed, detected or identified by a phenotypic orgenotypic aberration of LY6H genetic components or expression during thecourse or etiology of the disease or disorder. In this regard a LY6Hphenotypic aberration or determinant may, for example, compriseincreased or decreased levels of LY6H protein expression on one cellpopulation, e.g., a cancer cell population, or an immune cell population(such as a tumor infiltrating cell population), as compared to anothercell population, e.g., a normal cell population, or increased ordecreased LY6H protein expression on certain definable cell populations,or increased or decreased LY6H protein expression at an inappropriatephase or stage of a cell lifecycle. It will be appreciated that similarexpression patterns of genotypic determinants (e.g., mRNA transcriptionlevels) of LY6H may also be used to classify or detect LY6H associateddisorders. An “LY6H associated disorder,” as used herein, also includesa disorder characterized by infiltration of cells expressing LY6H, e.g.,LY6H expressing tumor and cancer cells. In one embodiment, a LY6Hassociated disorder is small cell lung cancer (SCLC). In one embodiment,a LY6H associated disorder is gastrointestinal stromal tumor (GIST). Inanother embodiment, a LY6H associated disorder is T cell acutelymphoblastic leukemia (T-ALL). In another embodiment, a LY6H associateddisorder is glioblastoma. In another embodiment, a LY6H associateddisorder is soft tissue sarcoma. In one embodiment, a LY6H associateddisorder is breast cancer. In one embodiment, a LY6H associated disorderis brain and CNS cancer. In one embodiment, a LY6H associated disorderis head and neck cancer. In one embodiment, a LY6H associated disorderis bladder cancer. In one embodiment, a LY6H associated disorder isrenal cancer. In another embodiment, a LY6H associated disorder isovarian cancer. In one embodiment, a LY6H associated disorder iseosophageal cancer. In another embodiment, a LY6H associated disorder isgastric cancer. In another embodiment, a LY6H associated disorder isprostrate cancer. In another embodiment, a LY6H associated disorder isuterine cancer. In another embodiment, a LY6H associated disorder iscolorectal cancer. In one embodiment, a LY6H associated disorder iscervical cancer. In another embodiment, a LY6H associated disorder isnon small cell lung cancer (NSCLC). In yet another embodiments, a LY6Hassociated disorder is endometrial cancer. In one embodiment, a LY6Hassociated disorder is pancreatic cancer. In another embodiment, a LY6Hassociated disorder is liver cancer.

In one embodiment, the SCLC is a classic SCLC. In another embodiment,the SCLC is a variant SCLC. “Classic SCLC” and “variant SCLCs” are wellknown to one of ordinary skill in the art. For example, see Broers etal., Cytometry, 9:426-431, 1988; Doyle et al., Cancer Res,49(23):6745-6751, 1989; Carney et al., Cancer Res., 45:2913-2923, 1985;and Koros et al., Lung Cancer, 7(4):225-234, 1991.

The term “cancer,” as used herein, is meant to refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include, but arenot limited to, breast cancer (Luminal A, TNBC, Ductal), prostatecancer, squamous cell tumors, squamous cell carcinoma (e.g., squamouscell lung cancer or squamous cell head and neck cancer), neuroendocrinetumors, urothelial cancer, vulvar cancer, mesothelioma, liver cancer,bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck,lung cancer, small cell lung cancer, non-small cell lung cancer,cutaneous or intraocular malignant melanoma, renal cancer, uterinecancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer,cancer of the anal region, stomach cancer, gastrointestinal stromaltumor (GIST), testicular cancer, uterine cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma,cancer of the esophagus, cancer of the small intestine, cancer of theendocrine system, cancer of the parathyroid gland, cancer of the adrenalgland, sarcoma of soft tissue, cancer of the urethra, cancer of thepenis, solid tumors of childhood, lymphocytic lymphoma, cancer of thebladder, cancer of the kidney or ureter, carcinoma of the renal pelvis,neoplasm of the central nervous system (CNS), primary CNS lymphoma,tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitaryadenoma, Kaposi's sarcoma, epidermoid cancer, environmentally inducedcancers including those induced by asbestos, hematologic malignanciesincluding, for example, multiple myeloma, B-cell lymphoma, Hodgkinlymphoma/primary mediastinal B-cell lymphoma, non-Hodgkin's lymphomas,acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoidleukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt'slymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosisfungoides, anaplastic large cell lymphoma, T-cell lymphoma, T cell acutelymphoblastic leukemia (T-ALL), and precursor T-lymphoblastic lymphoma,and any combinations of said cancers. PVNS, acute myeloid leukemia,adrenocortico carcinoma, ladder urothelial carcinoma, cervical squamouscell carcinoma, endocervical adenocarcinoma, diffuse large B celllymphoma, glioblastoma multiforme, chronic lymphocytic leukemia, brainlower grade glioma, head and neck squamous cell carcinoma,hepatocellular carcinoma, lung adenocarcinoma, large squamous cellcarcinoma, cutaneous melanoma, ovarial serous cystadenocarcinoma,gastric cancer, soft tissue sarcoma, testicular germ cell cancer,thymoma, thyroid carcinoma, uterine corpus endometrial carcinoma,uterine carcinosarcoma, kidney renal clear cell carcinoma, and kidneyrenal papillary cell carcinoma. The present invention is also applicableto treatment of metastatic cancers.

In one embodiment, an anti-LY6H ADC of the present invention can be usedto treat a cancer in a subject including, but not limited to Hodgkin'slymphoma, PVNS, acute myeloid leukemia, adrenocortico carcinoma, ladderurothelial carcinoma, breast cancer (Luminal A, TNBC, Ductal), cervicalsquamous cell carcinoma, endocervical adenocarcinoma, colorectaladenocarcinoma, diffuse large B cell lymphoma, non-hodgkin's lymphoma,glioblastoma multiforme, chronic lymphocytic leukemia, brain lower gradeglioma, head and neck squamous cell carcinoma, hepatocellular carcinoma,lung adenocarcinoma, small cell lung cancer, large squamous cellcarcinoma, cutaneous melanoma, ovarial serous cystadenocarcinoma,gastric cancer, soft tissue sarcoma, mesothelioma, pancreaticadenocarcinoma, testicular germ cell cancer, thymoma, thyroid carcinoma,uterine corpus endometrial carcinoma, uterine carcinosarcoma. kidneyrenal clear cell carcinoma, and kidney renal papillary cell carcinoma.

In one embodiment, the antibodies or ADCs of the invention areadministered to a patient having a cancer, including but not limited to,small cell lung cancer (SCLC), gastrointestinal stromal tumor (GIST), Tcell acute lymphoblastic leukemia (T-ALL), glioblastoma, and soft tissuesarcoma. In one embodiment, the antibodies or ADCs are administered to apatient having a classic SCLC. In another embodiment, the antibodies orADCs are administered to a patient having a variant SCLC.

In one embodiment, the antibodies or ADCs of the invention areadministered to a patient having a solid tumor, including an advancedsolid tumor. In one embodiment, the tumor expresses LY6H or containstumor infiltrating immune cells expressing LY6H. In another embodiment,the tumor does not express LY6H and/or does not contain tumorinfiltrating immune cells expressing LY6H. In another embodiment,administration of the antibodies of the invention to a patientupregulates an immune response in the patient. In another embodiment,administration of ADCs of the invention induce cell death of LY6Hexpressing cells.

The term “LY6H expressing tumor,” as used herein, refers to a tumorwhich expresses LY6H protein (including a tumor comprising tumorinfiltrating cells that express LY6H protein), such as a small cell lungcancer (SCLC) tumor, gastrointestinal stromal tumor (GIST), glioblastomatumor, and soft tissue sarcoma tumor. In one embodiment, LY6H expressionin a tumor is determined using immunohistochemical staining of tumorcell membranes, where any immunohistochemical staining above backgroundlevel in a tumor sample indicates that the tumor is a LY6H expressingtumor. In another embodiment, a LY6H expressing tumor, e.g., a smallcell lung cancer (SCLC) tumor, gastrointestinal stromal tumor (GIST),glioblastoma tumor, and soft tissue sarcoma tumor expressing LY6H, isidentified in a patient when greater than 1%, greater than 2%, greaterthan 3%, greater than 4%, greater than 5%, greater than 6%, greater than7%, greater than 8%, greater than 9%, greater than 10%, greater than15%, greater than 20%, greater than 25%, or greater than 30%, greaterthan 40%, greater than 50%, greater than 60%, greater than 70%, greaterthan 80%, greater than 90%, or more of the cells in a tumor sample arepositive for LY6H expression. In one embodiment, the LY6H expressingcells in the sample are tumor infiltrating immune cells. In anotherembodiment, LY6H positive expression is determined based on membranestaining as determined by, e.g., immunohistochemistry (IHC) analysis.

A LY6H expressing tumor is identified as having an “elevated level ofLY6H” or “expressing LY6H at an elevated level” when the level of LY6His higher than in tissue surrounding the cancer. In some embodiments, an“elevated level of LY6H” is one in which 5% or more of the cells in atumor sample have membrane staining. In some embodiments a “high level”in regard to LY6H is 5% or more staining, for example, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, or 100% of the cells in the tumor sample arestained. In some embodiments, the protein expression levels can bemeasured by IHC analysis. In another embodiment, the LY6H expressingcells in the sample are tumor infiltrating immune cells.

A LY6H expressing tumor is identified as having a “low level of LY6H” or“expressing LY6H at a low level” is one in which 5% or less of the cellsin a tumor sample have membrane staining In some embodiments a “lowlevel” in regard to LY6H is 5% or less staining, for example, 4.9, 4.5,4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1% or less of thecells in the tumor sample are stained. In some embodiments, the proteinexpression levels can be measured by IHC analysis. In anotherembodiment, the LY6H expressing cells in the sample are tumorinfiltrating immune cells.

A cell that expresses no LY6H can also be described as expressing a “lowlevel of LY6H”. Thus, the phrase “expresses a low level of LY6H”encompasses no LY6H expression. In some embodiments, a low level of LY6His within the background staining levels. In some embodiments, a samplethat is LY6H “negative” has no LY6H expression or a low level of LY6H.In some embodiments, LY6H staining is negative when no or less than 5%,4%, 3%, 2%, or 1% of the cells have membrane staining for LY6H.

As used herein, the term “tumor sample” refers to a tumor tissue or cellsample obtained from a tumor, e.g., including, but not limited to, asmall cell lung cancer (SCLC) tumor, gastrointestinal stromal tumor(GIST), glioblastoma tumor, and soft tissue sarcoma tumor. The samplecan include both tumor cells and tumor infiltrating cells, e.g., tumorinfiltrating immune cells.

As used herein, the term “non-cancer sample” or “normal sample” refersto a sample from a normal tissue (e.g., a lung tissue sample). In someembodiments, the non-cancer sample comes from the same subject, but isfrom a different part of the subject than that being tested. In someembodiments, the non-cancer sample is from a tissue area surrounding oradjacent to the cancer, e.g., small cell lung cancer (SCLC),gastrointestinal stromal tumor (GIST), T cell acute lymphoblasticleukemia (T-ALL), glioblastoma, and soft tissue sarcoma. In someembodiments, the non-cancer sample is not from the subject being tested,but is a sample from a subject known to have, or not to have, a disorderin question (for example, a particular cancer such as small cell lungcancer (SCLC), gastrointestinal stromal tumor (GIST), T cell acutelymphoblastic leukemia (T-ALL), glioblastoma, and soft tissue sarcoma orLY6H related disorder). In some embodiments, the non-cancer sample isfrom the same subject, but from a point in time before the subjectdeveloped cancer. In some embodiments, the reference sample is from abenign tumor or blood sample (for example, benign lung tumor sample),from the same or a different subject.

Methods for detecting expression of LY6H in a tumor are known in theart. For example, immunohistochemistry (IHC) analysis may be used toshow that LY6H is, for example, small cell lung cancer (SCLC) tissue,gastrointestinal stromal tumor (GIST) tissue, T cell acute lymphoblasticleukemia (T-ALL) cells, glioblastoma tissue, and soft tissue sarcomatissue.

The terms “overexpress,” “overexpression,” or “overexpressed”interchangeably refer to a gene that is transcribed or translated at adetectably greater level, usually in a cancer cell, in comparison to anormal cell. Overexpression therefore refers to both overexpression ofprotein and RNA (due to increased transcription, post transcriptionalprocessing, translation, post translational processing, alteredstability, and altered protein degradation), as well as localoverexpression due to altered protein traffic patterns (increasednuclear localization), and augmented functional activity, e.g., as in anincreased enzyme hydrolysis of substrate. Thus, overexpression refers toeither protein or RNA levels. Overexpression can also be by 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a normalcell or comparison cell. In certain embodiments, the anti-LY6Hantibodies or ADCs are used to treat tumors likely to overexpress LY6H.

The term “administering” as used herein is meant to refer to thedelivery of a substance (e.g., an anti-LY6H antibody or ADC) to achievea therapeutic objective (e.g., the treatment of an LY6H-associateddisorder or the inhibition or reduction of a tumor). Modes ofadministration may be parenteral, enteral and topical. Parenteraladministration is usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The term “combination therapy”, as used herein, refers to theadministration of two or more therapeutic substances, e.g., an anti-LY6Hantibody or ADC and an additional therapeutic agent. The additionaltherapeutic agent may be administered concomitant with, prior to, orfollowing the administration of the anti-LY6H antibody or ADC. In oneembodiment, the anti-LY6H antibodies or ADCs of the invention areadministered in combination with one or more immune checkpointinhibitors (e.g., one or more antibody or small molecule immunecheckpoint inhibitors) for the treatment of a cancer.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” refers to the amount of a drug, e.g., an antibody orADC, which is sufficient to reduce or ameliorate the severity and/orduration of a disorder, e.g., cancer, or one or more symptoms thereof,prevent the advancement of a disorder, cause regression of a disorder,prevent the recurrence, development, onset or progression of one or moresymptoms associated with a disorder, detect a disorder, or enhance orimprove the prophylactic or therapeutic effect(s) of another therapy(e.g., prophylactic or therapeutic agent). The effective amount of anantibody or ADC may, for example, inhibit tumor growth (e.g., inhibit anincrease in tumor volume), decrease tumor growth (e.g., decrease tumorvolume), reduce the number of cancer cells, and/or relieve to someextent one or more of the symptoms associated with the cancer. Theeffective amount may, for example, improve disease free survival (DFS),improve overall survival (OS), or decrease likelihood of recurrence.

Various aspects of the invention are described in further detail in thefollowing subsections.

II. Anti-LY6H Antibodies

One aspect disclosed herein provides humanized anti-LY6H antibodies, orantigen binding portions thereof. Another aspect disclosed hereinprovides human anti-LY6H antibodies, or antigen binding portionsthereof. Another aspect disclosed herein provides mouse anti-LY6Hantibodies, or antigen binding portions thereof. In one embodiment, theantibodies disclosed herein bind human LY6H. In another embodiment, theantibodies disclosed herein bind cynomolgus monkey LY6H. In anotherembodiment, the antibodies disclosed herein bind rhesus monkey LY6H. Inanother embodiment, the antibodies disclosed herein bind human LY6H oncells expressing LY6H, e.g., cancer cells, including, but not limitedto, small cell lung cancer (SCLC) cells, e.g., classic SCLC cells orvariant SCLC cells, gastrointestinal stromal tumor (GIST) cells, T cellacute lymphoblastic leukemia (T-ALL) cells, glioblastoma cells, and softtissue sarcoma cells. In another embodiment, the antibodies disclosedherein bind human LY6H expressed on tumor infiltrating cells, e.g.,tumor infiltrating immune cells. In another embodiment, the antibodiesdisclosed herein bind human LY6H expressed on tumor cells.

Another aspect disclosed herein features antibody drug conjugates (ADCs)comprising an anti-LY6H antibody described herein and at least onedrug(s). The antibodies or ADCs disclosed herein have characteristicsincluding, but not limited to, binding to human LY6H in vitro; bindinghuman LY6H on cells expressing LY6H in vivo, e.g., LY6H expressingcancer cells; binding human LY6H expressed on tumor infiltrating cells,e.g., tumor infiltrating immune cells; binding human LY6H expressed ontumor cells; regulating an immune response in vivo; undergoinginternalization in and inhibiting cells expressing LY6H, e.g., LY6Hexpressing tumor and cancer cells; inducing cell death in cellsexpressing LY6H, including, but not limited to, LY6H expressing tumorand cancer cells; inhibiting cancer cell invasion and metastasis;decreasing or inhibiting cancer, including, but not limited to, smallcell lung cancer (SCLC), e.g., classic SCLC or variant SCLC,gastrointestinal stromal tumor (GIST), T cell acute lymphoblasticleukemia (T-ALL), glioblastoma, and soft tissue sarcoma; and decreasingor inhibiting tumor cellular proliferation or tumor growth, or tumorinvasion and metastasis. ADCs disclosed herein, in particular, havecharacteristics including, but not limited to, inducing cell death incells expressing LY6H, e.g., cancer cells expressing LY6H. In oneembodiment, an anti-LY6H antibody or ADC disclosed herein is capable ofbeing internalized into a cell expressing LY6H.

In one embodiment, anti-LY6H antibodies are disclosed which have theability to bind to LY6H, as described in the Examples below.Collectively, the novel antibodies are referred to herein as “LY6Hantibodies.” In one embodiment, the anti-LY6H antibodies, ADCs, orantigen binding fragments thereof, are able to inhibit or decreaseprogression of cancer in vivo. In another embodiment, the anti-LY6Hantibodies, ADCs, or antigen binding fragments thereof, are able toinhibit or decrease tumor growth in vivo. The tumor can be a LY6Hnegative tumor or an LY6H expressing tumor. In various embodiments,anti-LY6H antibodies, ADCs, or antigen binding fragments thereof, arecapable of modulating a biological function of LY6H. In otherembodiments of the foregoing aspects, the anti-LY6H antibodies, ADCs, orantigen binding fragments thereof, bind LY6H on cells expressing LY6H,e.g., tumor and cancer cells expressing LY6H. Thus, the disclosureincludes anti-LY6H antibodies, ADCs, or antigen binding fragmentsthereof, that are effective at inhibiting or decreasing cancer or tumorgrowth.

In addition, the present inventors have shown that LY6H is expressed bysmall cell lung cancer (SCLC) cells (see Example 1), e.g., classic SCLCand/or variant SCLC. Increased expression of LY6H has been shown inmultiple cancer types (Luo, L. et al. Oncotarget 7(10): 11165-11193,2016). Accordingly, the anti-LY6H antibodies, ADCs, and antigen-bindingportions thereof, can be used for the treatment of cancers, including,but not limited to, small cell lung cancer (SCLC), gastrointestinalstromal tumor (GIST), T cell acute lymphoblastic leukemia (T-ALL),glioblastoma, and soft tissue sarcoma in a subject.

In one embodiment, greater than 1%, greater than 2%, greater than 3%,greater than 4%, greater than 5%, greater than 6%, greater than 7%,greater than 8%, greater than 9%, greater than 10%, greater than 15%,greater than 20%, greater than 25%, or greater than 30%, greater than40%, greater than 50%, greater than 60%, greater than 70%, greater than80%, greater than 90%, or more of the cells in a small cell lung cancer(SCLC) tumor sample are positive for LY6H expression. In anotherembodiment, a SCLC tumor sample has a high level of LY6H expression. Forexample, in one embodiment, at least 5% or more of the cells in a SCLCtumor sample have membrane staining. In another embodiment, a SCLC tumorsample obtained from the subject displays a low level of expression ofLY6H. The expression level of LY6H can be determined by any method knownin the art. For example, the expression level of LY6H can be determinedvia immunohistochemical analysis. In another embodiment, the SCLC hasbeen previously treated with another anti-cancer agent or anti-cancertherapy, e.g., a chemotherapy. In one embodiment, the SCLC is resistantto chemotherapy.

In one embodiment, greater than 1%, greater than 2%, greater than 3%,greater than 4%, greater than 5%, greater than 6%, greater than 7%,greater than 8%, greater than 9%, greater than 10%, greater than 15%,greater than 20%, greater than 25%, or greater than 30%, greater than40%, greater than 50%, greater than 60%, greater than 70%, greater than80%, greater than 90%, or more of the cells in a gastrointestinalstromal tumor (GIST) sample are positive for LY6H expression. In anotherembodiment, a GIST sample has a high level of LY6H expression. Forexample, in one embodiment, at least 5% or more of the cells in a GISTsample have membrane staining. In another embodiment, a GIST sampleobtained from the subject displays a low level of expression of LY6H.The expression level of LY6H can be determined by any method known inthe art. For example, the expression level of LY6H can be determined viaimmunohistochemical analysis. In another embodiment, the GIST has beenpreviously treated with another anti-cancer agent or anti-cancertherapy, e.g., a chemotherapy. In one embodiment, the GIST is resistantto chemotherapy.

In one embodiment, greater than 1%, greater than 2%, greater than 3%,greater than 4%, greater than 5%, greater than 6%, greater than 7%,greater than 8%, greater than 9%, greater than 10%, greater than 15%,greater than 20%, greater than 25%, or greater than 30%, greater than40%, greater than 50%, greater than 60%, greater than 70%, greater than80%, greater than 90%, or more of the cells in a sample, e.g., bloodsample, obtained from a subject having T cell acute lymphoblasticleukemia (T-ALL) are positive for LY6H expression. In anotherembodiment, a sample obtained from the subject having T-ALL has a highlevel of LY6H expression. For example, in one embodiment, at least 5% ormore of the cells in a sample obtained from the subject having T-ALLhave membrane staining. In another embodiment, a sample obtained fromthe subject having T-ALL displays a low level of expression of LY6H. Theexpression level of LY6H can be determined by any method known in theart. For example, the expression level of LY6H can be determined viaimmunohistochemical analysis. In another embodiment, the T-ALL has beenpreviously treated with another anti-cancer agent or anti-cancertherapy, e.g., a chemotherapy. In one embodiment, the T-ALL is resistantto chemotherapy.

In one embodiment, greater than 1%, greater than 2%, greater than 3%,greater than 4%, greater than 5%, greater than 6%, greater than 7%,greater than 8%, greater than 9%, greater than 10%, greater than 15%,greater than 20%, greater than 25%, or greater than 30%, greater than40%, greater than 50%, greater than 60%, greater than 70%, greater than80%, greater than 90%, or more of the cells in a glioblastoma tumorsample are positive for LY6H expression. In another embodiment, aglioblastoma tumor sample has a high level of LY6H expression. Forexample, in one embodiment, at least 5% or more of the cells in aglioblastoma tumor sample have membrane staining. In another embodiment,a glioblastoma tumor sample obtained from the subject displays a lowlevel of expression of LY6H. The expression level of LY6H can bedetermined by any method known in the art. For example, the expressionlevel of LY6H can be determined via immunohistochemical analysis. Inanother embodiment, the glioblastoma has been previously treated withanother anti-cancer agent or anti-cancer therapy, e.g., a chemotherapy.In one embodiment, the glioblastoma is resistant to chemotherapy.

In one embodiment, greater than 1%, greater than 2%, greater than 3%,greater than 4%, greater than 5%, greater than 6%, greater than 7%,greater than 8%, greater than 9%, greater than 10%, greater than 15%,greater than 20%, greater than 25%, or greater than 30%, greater than40%, greater than 50%, greater than 60%, greater than 70%, greater than80%, greater than 90%, or more of the cells in a soft tissue sarcomatumor sample are positive for LY6H expression. In another embodiment, asoft tissue sarcoma tumor sample has a high level of LY6H expression.For example, in one embodiment, at least 5% or more of the cells in asoft tissue sarcoma rumor sample have membrane staining. In anotherembodiment, a soft tissue sarcoma tumor sample obtained from the subjectdisplays a low level of expression of LY6H. The expression level of LY6Hcan be determined by any method known in the art. For example, theexpression level of LY6H can be determined via immunohistochemicalanalysis. In another embodiment, the soft tissue sarcoma has beenpreviously treated with another anti-cancer agent or anti-cancertherapy, e.g., a chemotherapy. In one embodiment, the soft tissuesarcoma is resistant to chemotherapy.

Antibodies having combinations of any of the aforementionedcharacteristics are contemplated as aspects of the disclosure. ADCs,described in more detail below, may also have any of the foregoingcharacteristics.

One aspect of the disclosure features an anti-human LY6H (anti-hLY6H)Antibody Drug Conjugate (ADC) comprising an anti-hLY6H antibodyconjugated to a drug via a linker Exemplary anti-LY6H antibodies (andsequences thereof) that can be used in the ADCs are described herein.

The anti-LY6H antibodies described herein provide the ADCs with theability to bind to LY6H such that the cytotoxic molecule attached to theantibody may be delivered to the LY6H-expressing cell, particularly aLY6H expressing cancer cell.

While the term “antibody” is used throughout, it should be noted thatantibody fragments (i.e., antigen-binding portions of an anti-LY6Hantibody) are also included in the disclosure and may be included in theembodiments (methods and compositions) described throughout. Forexample, an anti-LY6H antibody fragment may be conjugated to the drugs,as described herein. In certain embodiments, an anti-LY6H antibodybinding portion is a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linkedFv, an scFv, a single domain antibody, or a diabody.

Example 2 describes the generation of human monoclonal LY6H antibodiesagainst the extracellular domain (ECD) of human, monkey, rat, and mouseLY6H, identified herein as 6D11, 11D9, 23F3, 12G7, 22A5, 26F3, 35H10,1F8, 2B11, 24A10, 34H6, 10B10, 12G7_S54A, 12G7_N52Q, 6D11_41B4, and10B10_S54A. The heavy and light chain variable region amino acidsequences for the human antibodies are set forth in Table 6.

Thus, in one embodiment, the disclosure includes human anti-LY6Hantibodies, or antigen binding portions thereof, comprising a heavychain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1, 9, 17, 22, 27, 35, 39, 45, 53,54, 58, 60, 64, 66, 68, and 70; and a light chain variable regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 5, 13, 20, 26, 31, 37, 41, 49, and 56.

In one embodiment, the disclosure includes a human anti-LY6H antibody,or antigen binding portion thereof, comprising a Heavy Chain CDR set(CDR1, CDR2, and CDR3) selected from the group consisting of SEQ ID NOs:2, 3, and 4; SEQ ID NOs: 10, 11, and 12; SEQ ID NOs: 18, 11, and 19; SEQID NOs: 23, 24, and 25; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 28, 29,and 36; SEQ ID NOs: 28, 29, and 40; SEQ ID NOs: 46, 47, and 48; SEQ IDNOs: 46, 29, and 48; SEQ ID NOs: 28, 29 and 55; SEQ ID NOs: 28, 29, and59; SEQ ID NOs: 61, 62, and 63; SEQ ID NOs: 23, 65, and 25; SEQ ID NOs:23, 67, and 25; SEQ ID NOs: 2, 69, and 4; and SEQ ID NOs: 61, 71, and63; and a Light Chain CDR set (CDR1, CDR2, and CDR3) selected from thegroup consisting of SEQ ID NOs: 6, 7, and 8; SEQ ID NOs: 14, 15, and 16;SEQ ID NOs: 14, 15, and 21; SEQ ID NOs: 32, 33, and 34; SEQ ID NOs: 32,33, and 38; SEQ ID NOs: 42, 43, and 44; SEQ ID NOs: 50, 51, and 52; andSEQ ID NOs: 50, 51, and 57.

In one embodiment, an anti-LY6H antibody, or antigen binding portionthereof, is the human antibody 6D11. The 6D11 antibody comprises a heavychain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 3, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 2, and a light chain variable region comprising aCDR3 domain comprising the amino acid sequence of SEQ ID NO: 8, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 7, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 6. In furtherembodiments, disclosed herein is an antibody having a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 1 and alight chain variable region comprising the amino acid sequence of SEQ IDNO: 5.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 1, or a sequence having at least 90%, 95%, 96%, 97%,98%, or 99% identity to SEQ ID NO: 1, and/or a light chain comprising anamino acid sequence set forth in SEQ ID NO: 5, or a sequence having atleast 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5.

In one embodiment, the disclosure features an anti-LY6H antibody, orantigen binding portion thereof, which is the human antibody 11D9. The11D9 antibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 10, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 16, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 15, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 14. In further embodiments, the disclosureprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 9 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 13.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 9, or a sequence having at least 90%, 95%, 96%, 97%,98%, or 99% identity to SEQ ID NO: 9, and/or a light chain comprising anamino acid sequence set forth in SEQ ID NO: 13, or a sequence having atleast 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13.

In one embodiment, the disclosure features an anti-LY6H antibody, orantigen binding portion thereof, which is the human antibody 23F3. The23F3 antibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 19, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 18, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 21, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 15, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 14. In further embodiments, the disclosureprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 17 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 20.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 17, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 17, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 20, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 20.

In one embodiment, the disclosure features an anti-LY6H antibody, orantigen binding portion thereof, which is the human antibody 12G7. The12G7 antibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 25, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 24, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 23, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6. In further embodiments, the disclosureprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 22 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 26.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 22, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 22, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 26, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 26.

In one embodiment, an anti-LY6H antibody, or antigen binding portionthereof, is the human antibody 22A5. The 22A5 antibody comprises a heavychain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 30, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 29, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 28, and a light chain variable region comprisinga CDR3 domain comprising the amino acid sequence of SEQ ID NO: 34, aCDR2 domain comprising the amino acid sequence of SEQ ID NO: 33, and aCDR1 domain comprising the amino acid sequence of SEQ ID NO: 32. Infurther embodiments, disclosed herein is an antibody having a heavychain variable region comprising the amino acid sequence of SEQ ID NO:27 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 31.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 27, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 27, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 31, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 31.

In one embodiment, an anti-LY6H antibody, or antigen binding portionthereof, is the human antibody 26F3. The 26F3 antibody comprises a heavychain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 36, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 29, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 28, and a light chain variable region comprisinga CDR3 domain comprising the amino acid sequence of SEQ ID NO: 38, aCDR2 domain comprising the amino acid sequence of SEQ ID NO: 33, and aCDR1 domain comprising the amino acid sequence of SEQ ID NO: 32. Infurther embodiments, disclosed herein is an antibody having a heavychain variable region comprising the amino acid sequence of SEQ ID NO:35 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 37.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 35, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 35, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 37, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 37.

In one embodiment, an anti-LY6H antibody, or antigen binding portionthereof, is the human antibody 35H10. The 35H10 antibody comprises aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 40, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 29, and a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 28, and a light chain variable regioncomprising a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 44, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:43, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:42. In further embodiments, disclosed herein is an antibody having aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 39 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 41.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 39, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 39, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 41, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 41.

In one embodiment, an anti-LY6H antibody, or antigen binding portionthereof, is the human antibody 1F8. The 1F81 antibody comprises a heavychain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 48, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 47, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 46, and a light chain variable region comprisinga CDR3 domain comprising the amino acid sequence of SEQ ID NO: 52, aCDR2 domain comprising the amino acid sequence of SEQ ID NO: 51, and aCDR1 domain comprising the amino acid sequence of SEQ ID NO: 50. Infurther embodiments, disclosed herein is an antibody having a heavychain variable region comprising the amino acid sequence of SEQ ID NO:45 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 49.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 45, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 45, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 49, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 49.

In one embodiment, an anti-LY6H antibody, or antigen binding portionthereof, is the human antibody 2B11. The 2B11 antibody comprises a heavychain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 48, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 29, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 46, and a light chain variable region comprisinga CDR3 domain comprising the amino acid sequence of SEQ ID NO: 52, aCDR2 domain comprising the amino acid sequence of SEQ ID NO: 51, and aCDR1 domain comprising the amino acid sequence of SEQ ID NO: 50. Infurther embodiments, disclosed herein is an antibody having a heavychain variable region comprising the amino acid sequence of SEQ ID NO:53 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 49.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 53, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 53, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 49, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 49.

In one embodiment, an anti-LY6H antibody, or antigen binding portionthereof, is the human antibody 24A10. The 24A10 antibody comprises aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 55, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 29, and a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 28, and a light chain variable regioncomprising a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 57, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:51, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:50. In further embodiments, disclosed herein is an antibody having aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 54 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 56.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 54, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 54, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 56, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 56.

In one embodiment, an anti-LY6H antibody, or antigen binding portionthereof, is the human antibody 34H6. The 34H6 antibody comprises a heavychain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 59, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 29, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 28, and a light chain variable region comprisinga CDR3 domain comprising the amino acid sequence of SEQ ID NO: 57, aCDR2 domain comprising the amino acid sequence of SEQ ID NO: 51, and aCDR1 domain comprising the amino acid sequence of SEQ ID NO: 50. Infurther embodiments, disclosed herein is an antibody having a heavychain variable region comprising the amino acid sequence of SEQ ID NO:58 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 56.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 58, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 58, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 56, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 56.

In one embodiment, an anti-LY6H antibody, or antigen binding portionthereof, is the human antibody 10B10. The 10B10 antibody comprises aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 63, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 62, and a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 61, and a light chain variable regioncomprising a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 7,and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 6. Infurther embodiments, disclosed herein is an antibody having a heavychain variable region comprising the amino acid sequence of SEQ ID NO:60 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 26.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 60, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 60, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 26, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 26.

In one embodiment, an anti-LY6H antibody, or antigen binding portionthereof, is the human antibody 12G7_S54A. The 12G7_S54A antibodycomprises a heavy chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 25, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 65, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 23, and a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6. In further embodiments, disclosed herein is anantibody having a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 64 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 26.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 64, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 64, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 26, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 26.

In one embodiment, an anti-LY6H antibody, or antigen binding portionthereof, is the human antibody 12G7_N52Q. The 12G7_N52Q antibodycomprises a heavy chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 25, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 67, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 23, and a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6. In further embodiments, disclosed herein is anantibody having a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 66 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 26.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 66, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 66, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 26, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 26.

In one embodiment, an anti-LY6H antibody, or antigen binding portionthereof, is the human antibody 6D11_41B4. The 6D11_41B4 antibodycomprises a heavy chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 4, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 69, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 2, and a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6. In further embodiments, disclosed herein is anantibody having a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 68 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 5.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 68, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 68, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 5, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 5.

In one embodiment, an anti-LY6H antibody, or antigen binding portionthereof, is the human antibody 10B10_S54A. The 10B10_S54A antibodycomprises a heavy chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 63, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 71, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 61 and a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6. In further embodiments, disclosed herein is anantibody having a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 70 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 26.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 70, or a sequence having at least 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 70, and/or a light chaincomprising an amino acid sequence set forth in SEQ ID NO: 26, or asequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 26.

In some embodiments, an anti-LY6H antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequencedescribed herein, or a sequence having at least 90%, 95%, 96%, 97%, 98%,or 99% identity to a sequence deacribed herein, and/or a light chaincomprising an amino acid sequence described herein, or a sequence havingat least 90%, 95%, 96%, 97%, 98%, or 99% identity to a sequencedescribed herein.

The foregoing anti-LY6H antibody CDR sequences establish a novel familyof LY6H binding proteins, isolated in accordance with this disclosure,and comprising antigen binding polypeptides that include the CDRsequences listed in Table 6.

To generate and to select CDRs having preferred LY6H binding and/orneutralizing activity with respect to hLY6H, standard methods known inthe art for generating antibodies, or antigen binding portions thereof,and assessing the LY6H binding and/or neutralizing characteristics ofthose antibodies, or antigen binding portions thereof, may be used,including but not limited to those specifically described herein.

In certain embodiments, the antibody comprises a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgDconstant region. In certain embodiments, the anti-LY6H antibody, orantigen binding portion thereof, comprises a heavy chain immunoglobulinconstant domain selected from the group consisting of a human IgGconstant domain, a human IgM constant domain, a human IgE constantdomain, and a human IgA constant domain. In further embodiments, theantibody, or antigen binding portion thereof, has an IgG1 heavy chainconstant region, an IgG2 heavy chain constant region, an IgG3 constantregion, or an IgG4 heavy chain constant region. Preferably, the heavychain constant region is an IgG1 heavy chain constant region or an IgG4heavy chain constant region. Furthermore, the antibody can comprise alight chain constant region, either a kappa light chain constant regionor a lambda light chain constant region. Preferably, the antibodycomprises a kappa light chain constant region. Alternatively, theantibody portion can be, for example, a Fab fragment or a single chainFv fragment.

In certain embodiments, the anti-LY6H antibody binding portion is a Fab,a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, an scFv, a single domainantibody, or a diabody.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, is a multispecific antibody, e.g. a bispecificantibody.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 1 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 5.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 9 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 13.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 17 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 20.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 22 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 26.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 27 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 31.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 35 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 37.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 39 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 41.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 45 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 49.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 53 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 49.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 54 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 56.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 58 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 56.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 60 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 26.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 64 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 26.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 66 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 26.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 68 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 5.

In certain embodiments, the anti-LY6H antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 70 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 26.

Replacements of amino acid residues in the Fc portion to alter antibodyeffector function are have been described (Winter, et al. U.S. Pat. Nos.5,648,260 and 5,624,821, incorporated by reference herein). The Fcportion of an antibody mediates several important effector functionse.g. cytokine induction, ADCC, phagocytosis, complement dependentcytotoxicity (CDC) and half-life/clearance rate of antibody andantigen-antibody complexes. In some cases these effector functions aredesirable for therapeutic antibody but in other cases might beunnecessary or even deleterious, depending on the therapeuticobjectives. Certain human IgG isotypes, particularly IgG1 and IgG3,mediate ADCC and CDC via binding to FcγRs and complement C1q,respectively. Neonatal Fc receptors (FcRn) are the critical componentsdetermining the circulating half-life of antibodies. In still anotherembodiment at least one amino acid residue is replaced in the constantregion of the antibody, for example the Fc region of the antibody, suchthat effector functions of the antibody are altered.

One embodiment includes a labeled anti-LY6H antibody, or antibodyportion thereof, where the antibody is derivatized or linked to one ormore functional molecule(s) (e.g., another peptide or protein). Forexample, a labeled antibody can be derived by functionally linking anantibody or antibody portion of the disclosure (by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), a detectable agent, a pharmaceutical agent, aprotein or peptide that can mediate the association of the antibody orantibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag), and/or a cytotoxic or therapeutic agentselected from the group consisting of a mitotic inhibitor, an antitumorantibiotic, an immunomodulating agent, a vector for gene therapy, analkylating agent, an antiangiogenic agent, an antimetabolite, aboron-containing agent, a chemoprotective agent, a hormone, anantihormone agent, a corticosteroid, a photoactive therapeutic agent, anoligonucleotide, a radionuclide agent, a topoisomerase inhibitor, atyrosine kinase inhibitor, a radiosensitizer, and a combination thereof.

Useful detectable agents with which an antibody or antibody portionthereof, may be derivatized include fluorescent compounds. Exemplaryfluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

In one embodiment, the antibody is conjugated to an imaging agent.Examples of imaging agents that may be used in the compositions andmethods described herein include, but are not limited to, a radiolabel(e.g., indium), an enzyme, a fluorescent label, a luminescent label, abioluminescent label, a magnetic label, and biotin.

In one embodiment, the antibodies or ADCs are linked to a radiolabel,such as, but not limited to, indium (¹¹¹In). ¹¹¹Indium may be used tolabel the antibodies and ADCs described herein for use in identifyingLY6H positive tumors. In a certain embodiment, anti-LY6H antibodies (orADCs) described herein are labeled with ¹¹¹I via a bifunctional chelatorwhich is a bifunctional cyclohexyl diethylenetriaminepentaacetic acid(DTPA) chelate (see U.S. Pat. Nos. 5,124,471; 5,434,287; and 5,286,850,each of which is incorporated herein by reference).

Another embodiment of the disclosure provides a glycosylated bindingprotein wherein the anti-LY6H antibody or antigen binding portionthereof comprises one or more carbohydrate residues. Nascent in vivoprotein production may undergo further processing, known aspost-translational modification. In particular, sugar (glycosyl)residues may be added enzymatically, a process known as glycosylation.The resulting proteins bearing covalently linked oligosaccharide sidechains are known as glycosylated proteins or glycoproteins. Antibodiesare glycoproteins with one or more carbohydrate residues in the Fcdomain, as well as the variable domain. Carbohydrate residues in the Fcdomain have important effect on the effector function of the Fc domain,with minimal effect on antigen binding or half-life of the antibody (R.Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16). In contrast,glycosylation of the variable domain may have an effect on the antigenbinding activity of the antibody. Glycosylation in the variable domainmay have a negative effect on antibody binding affinity, likely due tosteric hindrance (Co, M. S., et al., Mol. Immunol. (1993) 30:1361-1367),or result in increased affinity for the antigen (Wallick, S. C., et al.,Exp. Med. (1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991)10:2717-2723).

One aspect of the disclosure is directed to generating glycosylationsite mutants in which the O- or N-linked glycosylation site of thebinding protein has been mutated. One skilled in the art can generatesuch mutants using standard well-known technologies. Glycosylation sitemutants that retain the biological activity, but have increased ordecreased binding activity, are another object of the disclosure.

In still another embodiment, the glycosylation of the anti-LY6H antibodyor antigen binding portion is modified. For example, an aglycoslatedantibody can be made (i.e., the antibody lacks glycosylation).Glycosylation can be altered to, for example, increase the affinity ofthe antibody for antigen. Such carbohydrate modifications can beaccomplished by, for example, altering one or more sites ofglycosylation within the antibody sequence. For example, one or moreamino acid substitutions can be made that result in elimination of oneor more variable region glycosylation sites to thereby eliminateglycosylation at that site. Such aglycosylation may increase theaffinity of the antibody for antigen. Such an approach is described infurther detail in PCT Publication WO2003016466A2, and U.S. Pat. Nos.5,714,350 and 6,350,861, each of which is incorporated herein byreference in its entirety.

Additionally or alternatively, a modified anti-LY6H antibody can be madethat has an altered type of glycosylation, such as a hypofucosylatedantibody having reduced amounts of fucosyl residues or an antibodyhaving increased bisecting GlcNAc structures. Such altered glycosylationpatterns have been demonstrated to increase the ADCC ability ofantibodies. Such carbohydrate modifications can be accomplished by, forexample, expressing the antibody in a host cell with alteredglycosylation machinery. Cells with altered glycosylation machinery havebeen described in the art and can be used as host cells in which toexpress recombinant antibodies to thereby produce an antibody withaltered glycosylation. See, for example, Shields, R. L. et al. (2002) J.Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1,as well as, European Patent No: EP 1,176,195; PCT Publications WO03/035835; WO 99/54342 80, each of which is incorporated herein byreference in its entirety.

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (e.g.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful may include, but are not limited to,glucose, galactose, mannose, fucose, n-acetylglucosamine and sialicacid. Preferably the glycosylated binding protein comprises glycosylresidues such that the glycosylation pattern is human.

Differing protein glycosylation may result in differing proteincharacteristics. For instance, the efficacy of a therapeutic proteinproduced in a microorganism host, such as yeast, and glycosylatedutilizing the yeast endogenous pathway may be reduced compared to thatof the same protein expressed in a mammalian cell, such as a CHO cellline. Such glycoproteins may also be immunogenic in humans and showreduced half-life in vivo after administration. Specific receptors inhumans and other animals may recognize specific glycosyl residues andpromote the rapid clearance of the protein from the bloodstream. Otheradverse effects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using recombinant techniques, apractitioner may generate antibodies or antigen binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (U.S. patent PublicationNos. 20040018590 and 20020137134 and PCT publication WO2005100584 A2).

Antibodies may be produced by any of a number of techniques. Forexample, expression from host cells, wherein expression vector(s)encoding the heavy and light chains is (are) transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. Although it ispossible to express antibodies in either prokaryotic or eukaryotic hostcells, expression of antibodies in eukaryotic cells is preferable, andmost preferable in mammalian host cells, because such eukaryotic cells(and in particular mammalian cells) are more likely than prokaryoticcells to assemble and secrete a properly folded and immunologicallyactive antibody.

Preferred mammalian host cells for expressing the recombinant antibodiesdisclosed herein include Chinese Hamster Ovary (CHO cells) (includingdhfr− CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl.Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g.,as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of thedisclosure. For example, it may be desirable to transfect a host cellwith DNA encoding functional fragments of either the light chain and/orthe heavy chain of an antibody. Recombinant DNA technology may also beused to remove some, or all, of the DNA encoding either or both of thelight and heavy chains that is not necessary for binding to the antigensof interest. The molecules expressed from such truncated DNA moleculesare also encompassed by the antibodies of the disclosure. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are an antibody of the disclosure and the other heavy and lightchain are specific for an antigen other than the antigens of interest bycrosslinking an antibody of the disclosure to a second antibody bystandard chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen binding portion thereof of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into CHO cell lines with deletions inthe dihydrofolate reductase (DHFR) or glutamine synthesis (GS) genes arewhich are suitable for generating stable cell lines for expression ofthese antibodies.

In another system for recombinant expression of an antibody, or antigenbinding portion thereof, a recombinant expression vector encoding boththe antibody heavy chain and the antibody light chain is introduced intodhfr-CHO cells by calcium phosphate-mediated transfection.

Within the recombinant expression vector, the antibody heavy and lightchain genes are each operatively linked to CMV enhancer/AdMLP promoterregulatory elements to drive high levels of transcription of the genes.The recombinant expression vector also carries a DHFR gene, which allowsfor selection of CHO cells that have been transfected with the vectorusing methotrexate selection/amplification. The selected transformanthost cells are cultured to allow for expression of the antibody heavyand light chains and intact antibody is recovered from the culturemedium. Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody from theculture medium. Still further the disclosure provides a method ofsynthesizing a recombinant antibody by culturing a host cell in asuitable culture medium until a recombinant antibody is synthesized.Recombinant antibodies may be produced using nucleic acid moleculescorresponding to the amino acid sequences disclosed herein. In oneembodiment, the nucleic acid molecules set forth in SEQ ID NOs: 97-122are used in the production of a recombinant antibody. The method canfurther comprise isolating the recombinant antibody from the culturemedium.

III. Anti-LY6H Antibody Drug Conjugates (ADCs)

Anti-LY6H antibodies described herein may be conjugated to a drug moietyto form an anti-LY6H Antibody Drug Conjugate (ADC). Antibody-drugconjugates (ADCs) may increase the therapeutic efficacy of antibodies intreating disease, e.g., cancer, due to the ability of the ADC toselectively deliver one or more drug moiety(s) to target tissues orcells, e.g., LY6H expressing tumors or LY6H expressing cells, such as,LY6H expressing cancer cells. Thus, in certain embodiments, thedisclosure provides anti-LY6H ADCs for therapeutic use, e.g., treatmentof cancer.

Anti-LY6H ADCs comprise an anti-LY6H antibody, i.e., an antibody thatspecifically binds to LY6H, linked to one or more drug moieties. Thespecificity of the ADC is defined by the specificity of the antibody,i.e., anti-LY6H. In one embodiment, an anti-LY6H antibody is linked toone or more cytotoxic drug(s) which is delivered internally to a cancercell expressing LY6H.

Examples of drugs that may be used in the anti-LY6H ADCs are providedbelow, as are linkers that may be used to conjugate the antibody and theone or more drug(s). The terms “drug,” “agent,” and “drug moiety” areused interchangeably herein. The terms “linked” and “conjugated” arealso used interchangeably herein and indicate that the antibody andmoiety are covalently linked.

In some embodiments, the ADC has the following formula (formula I):

Ab-(L-D)n  (I)

wherein Ab an anti-LY6H antibody described herein, and (L-D) is aLinker-Drug moiety. The Linker-Drug moiety is made of L- which is aLinker, and -D, which is a drug moiety having, for example, cytostatic,cytotoxic, or otherwise therapeutic activity against a target cell,e.g., a cell expressing LY6H; and n is an integer from 1 to 20. In someembodiments, n ranges from 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, or is 1. The DAR of an ADC is equivalent to the “n” referredto in Formula I.

Additional details regarding drugs (D of Formula I) and linkers (L ofFormula I) that may be used in the ADCs, as well as alternative ADCstructures, are described below.

A. Anti-LY6H ADCs: Exemplary Drugs for Conjugation

Anti-LY6H antibodies may be used in ADCs to target one or more drug(s)to a cell of interest, e.g., a cell expressing LY6H. The anti-LY6H ADCsdisclosed herein provide a targeted therapy that may, for example,reduce the side effects often seen with anti-cancer therapies, as theone or more drug(s) is delivered to a specific cell. In one embodiment,the drug used in an ADC is saporin. In another embodiment, the drug usedin an ADC is dacarbazine. In another embodiment, the drug used in an ADCis carboplatin.

Examples of drugs that may be used in ADCs, i.e., drugs that may beconjugated to the anti-LY6H antibodies, are provided below, and includemitotic inhibitors, antitumor antibiotics, immunomodulating agents, genetherapy vectors, alkylating agents, antiangiogenic agents,antimetabolites, boron-containing agents, chemoprotective agents,hormone agents, glucocorticoids, photoactive therapeutic agents,oligonucleotides, radioactive isotopes, radiosensitizers, topoisomeraseinhibitors, tyrosine kinase inhibitors, and combinations thereof.

1. Mitotic Inhibitors

In one aspect, anti-LY6H antibodies may be conjugated to one or moremitotic inhibitor(s) to form an ADC for the treatment of cancer. Theterm “mitotic inhibitor”, as used herein, refers to a cytotoxic and/ortherapeutic agent that blocks mitosis or cell division, a biologicalprocess particularly important to cancer cells. A mitotic inhibitordisrupts microtubules such that cell division is prevented, often byeffecting microtubule polymerization (e.g., inhibiting microtubulepolymerization) or microtubule depolymerization (e.g., stabilizing themicrotubule cytoskeleton against depolymrization). Thus, in oneembodiment, an anti-LY6H antibody of the invention is conjugated to oneor more mitotic inhibitor(s) that disrupts microtubule formation byinhibiting tubulin polymerization. In another embodiment, an anti-LY6Hantibody of the invention is conjugated to one or more mitoticinhibitor(s) that stabilizes the microtubule cytoskeleton fromdeploymerization. In one embodiment, the mitotic inhibitor used in theADCs of the invention is Ixempra (ixabepilone). Examples of mitoticinhibitors that may be used in the anti-LY6H ADCs of the invention areprovided below. Included in the genus of mitotic inhibitors areauristatins, described below.

a. Dolastatins

The anti-LY6H antibodies of the invention may be conjugated to at leastone dolastatin to form an ADC. Dolastatins are short peptidic compoundsisolated from the Indian Ocean sea hare Dolabella auricularia (seePettit et al., J. Am. Chem. Soc., 1976, 98, 4677). Examples ofdolastatins include dolastatin 10 and dolatstin 15. Dolastatin 15, aseven-subunit depsipeptide derived from Dolabella auricularia, and is apotent antimitotic agent structurally related to the antitubulin agentdolastatin 10, a five-subunit peptide obtained from the same organism.Thus, in one embodiment, the anti-LY6H ADC of the invention comprises ananti-LY6H antibody, as described herein, and at least one dolastatin.Auristatins are synthetic derivatives of dolastatin 10.

b. Auristatins

Anti-LY6H antibodies may be conjugated to at least one auristatin.Auristatins represent a group of dolastatin analogs that have generallybeen shown to possess anticancer activity by interfering withmicrotubule dynamics and GTP hydrolysis, thereby inhibiting cellulardivision. For example, Auristatin E (U.S. Pat. No. 5,635,483) is asynthetic analogue of the marine natural product dolastatin 10, acompound that inhibits tubulin polymerization by binding to the samesite on tubulin as the anticancer drug vincristine (G. R. Pettit, Prog.Chem. Org. Nat. Prod, 70: 1-79 (1997)). Dolastatin 10, auristatin PE,and auristatin E are linear peptides having four amino acids, three ofwhich are unique to the dolastatin class of compounds. Exemplaryembodiments of the auristatin subclass of mitotic inhibitors include,but are not limited to, monomethyl auristatin D (MMAD or auristatin Dderivative), monomethyl auristatin E (MMAE or auristatin E derivative),monomethyl auristatin F (MMAF or auristatin F derivative), auristatin Fphenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), and5-benzoylvaleric acid-AE ester (AEVB). The synthesis and structure ofauristatin derivatives are described in U.S. Patent ApplicationPublication Nos. 2003-0083263, 2005-0238649 and 2005-0009751;International Patent Publication No. WO 04/010957, International PatentPublication No. WO 02/088172, and U.S. Pat. Nos. 6,323,315; 6,239,104;6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902;5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036;5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414,each of which is incorporated by reference herein.

In one embodiment, anti-LY6H antibodies are conjugated to at least oneMMAE (monomethyl auristatin E). Monomethyl auristatin E (MMAE, vedotin)inhibits cell division by blocking the polymerization of tubulin.Because of its super toxicity, it also cannot be used as a drug itself.In recent cancer therapy developments, it is linked to a monoclonalantibody (mAb) that recognizes a specific marker expression in cancercells and directs MMAE to the cancer cells. In one embodiment, thelinker linking MMAE to the anti-LY6H antibody is stable in extracellularfluid (i.e., the medium or environment that is external to cells), butis cleaved by cathepsin once the ADC has bound to the specific cancercell antigen and entered the cancer cell, thus releasing the toxic MMAEand activating the potent anti-mitotic mechanism.

The structure of MMAE is provided below.

In one embodiment, the antibody is coupled to a single drug and,therefore, has a DAR of 1. In certain embodiments, the ADC will have aDAR of 2 to 8, 2 to 6, or 2 to 4.

c. Maytansinoids

The anti-LY6H antibodies of the invention may be conjugated to at leastone maytansinoid to form an ADC. Maytansinoids are potent antitumoragents that were originally isolated from members of the higher plantfamilies Celastraceae, Rhamnaceae, and Euphorbiaceae, as well as somespecies of mosses (Kupchan et al, J. Am. Chem. Soc. 94:1354-1356 [1972];Wani et al, J. Chem. Soc. Chem. Commun. 390: [1973]; Powell et al, J.Nat. Prod. 46:660-666 [1983]; Sakai et al, J. Nat. Prod. 51:845-850[1988]; and Suwanborirux et al, Experientia 46:117-120 [1990]). Evidencesuggests that maytansinoids inhibit mitosis by inhibiting polymerizationof the microtubule protein tubulin, thereby preventing formation ofmicrotubules (see, e.g., U.S. Pat. No. 6,441,163 and Remillard et al.,Science, 189, 1002-1005 (1975)). Maytansinoids have been shown toinhibit tumor cell growth in vitro using cell culture models, and invivo using laboratory animal systems. Moreover, the cytotoxicity ofmaytansinoids is 1,000-fold greater than conventional chemotherapeuticagents, such as, for example, methotrexate, daunorubicin, andvincristine (see, e.g., U.S. Pat. No. 5,208,020).

Maytansinoids to include maytansine, maytansinol, C-3 esters ofmaytansinol, and other maytansinol analogues and derivatives (see, e.g.,U.S. Pat. Nos. 5,208,020 and 6,441,163, each of which is incorporated byreference herein). C-3 esters of maytansinol can be naturally occurringor synthetically derived. Moreover, both naturally occurring andsynthetic C-3 maytansinol esters can be classified as a C-3 ester withsimple carboxylic acids, or a C-3 ester with derivatives ofN-methyl-L-alanine, the latter being more cytotoxic than the former.Synthetic maytansinoid analogues are described in, for example, Kupchanet al., J. Med. Chem., 21, 31-37 (1978).

Suitable maytansinoids for use in ADCs of the invention can be isolatedfrom natural sources, synthetically produced, or semi-syntheticallyproduced. Moreover, the maytansinoid can be modified in any suitablemanner, so long as sufficient cytotoxicity is preserved in the ultimateconjugate molecule. In this regard, maytansinoids lack suitablefunctional groups to which antibodies can be linked A linking moietydesirably is utilized to link the maytansinoid to the antibody to formthe conjugate, and is described in more detail in the linker sectionbelow. The structure of an exemplary maytansinoid, mertansine (DM1), isprovided below.

Representative examples of maytansinoids include, but are not limited,to DM1 (N²′-deacetyl-N²′-(3-mercapto-1-oxopropyl)-maytansine; alsoreferred to as mertansine, drug maytansinoid 1; ImmunoGen, Inc.; seealso Chari et al. (1992) Cancer Res 52:127), DM2, DM3(N²′-deacetyl-N²′-(4-mercapto-1-oxopentyl)-maytansine), DM4(4-methyl-4-mercapto-1-oxopentyl)-maytansine), and maytansinol (asynthetic maytansinoid analog). Other examples of maytansinoids aredescribed in U.S. Pat. No. 8,142,784, incorporated by reference herein.

Ansamitocins are a group of maytansinoid antibiotics that have beenisolated from various bacterial sources. These compounds have potentantitumor activities. Representative examples include, but are notlimited to ansamitocin P1, ansamitocin P2, ansamitocin P3, andansamitocin P4.

In one embodiment of the invention, an anti-LY6H antibody is conjugatedto at least one DM1. In one embodiment, an anti-LY6H antibody isconjugated to at least one DM2. In one embodiment, an anti-LY6H antibodyis conjugated to at least one DM3. In one embodiment, an anti-LY6Hantibody is conjugated to at least one DM4.

2. Antitumor Antibiotics

Anti-LY6H antibodies may be conjugated to one or more antitumorantibiotic(s) for the treatment of cancer. As used herein, the term“antitumor antibiotic” means an antineoplastic drug that blocks cellgrowth by interfering with DNA and is made from a microorganism. Often,antitumor antibiotics either break up DNA strands or slow down or stopDNA synthesis. Examples of antitumor antibiotics that may be included inthe anti-LY6H ADCs include, but are not limited to, actinomycines (e.g.,pyrrolo[2,1-c][1,4]benzodiazepines), anthracyclines, calicheamicins, andduocarmycins. In addition to the foregoing, additional antitumorantibiotics that may be used in the anti-LY6H ADCs include bleomycin(Blenoxane, Bristol-Myers Squibb), mitomycin, and plicamycin (also knownas mithramycin).

3. Immunomodulating Agents

In one aspect, anti-LY6H antibodies may be conjugated to at least oneimmunomodulating agent. As used herein, the term “immunomodulatingagent” refers to an agent that can stimulate or modify an immuneresponse. In one embodiment, an immunomodulating agent is animmunostimuator which enhances a subject's immune response. In anotherembodiment, an immunomodulating agent is an immunosuppressant whichprevents or decreases a subject's immune response. An immunomodulatingagent may modulate myeloid cells (monocytes, macrophages, dendriticcells, megakaryocytes and granulocytes) or lymphoid cells (T cells, Bcells and natural killer (NK) cells) and any further differentiated cellthereof. Representative examples include, but are not limited to,bacillus calmette-guerin (BCG) and levamisole (Ergamisol). Otherexamples of immunomodulating agents that may be used in the ADCsinclude, but are not limited to, cancer vaccines, and cytokines.

As used herein, the term “cancer vaccine” refers to a composition (e.g.,a tumor antigen and a cytokine) that elicits a tumor-specific immuneresponse. The response is elicited from the subject's own immune systemby administering the cancer vaccine, or, in the case of the instantdisclosure, administering an ADC comprising an anti-LY6H antibody and acancer vaccine. In preferred embodiments, the immune response results inthe eradication of tumor cells in the body (e.g., primary or metastatictumor cells). The use of cancer vaccines generally involves theadministration of a particular antigen or group of antigens that are,for example, present on the surface a particular cancer cell, or presenton the surface of a particular infectious agent shown to facilitatecancer formation. In some embodiments, the use of cancer vaccines is forprophylactic purposes, while in other embodiments, the use is fortherapeutic purposes. Non-limiting examples of cancer vaccines that maybe used in the anti-LY6H ADCs include, recombinant bivalent humanpapillomavirus (HPV) vaccine types 16 and 18 vaccine (Cervarix,GlaxoSmithKline), recombinant quadrivalent human papillomavirus (HPV)types 6, 11, 16, and 18 vaccine (Gardasil, Merck & Company), andsipuleucel-T (Provenge, Dendreon). Thus, in one embodiment, theanti-LY6H antibody is conjugated to at least one cancer vaccine that iseither an immunostimulator or is an immunosuppressant.

The anti-LY6H antibodies may be conjugated to at least one cytokine. Theterm “cytokine” generally refers to proteins released by one cellpopulation which act on another cell as intercellular mediators.Cytokines directly stimulate immune effector cells and stromal cells atthe tumor site and enhance tumor cell recognition by cytotoxic effectorcells (Lee and Margolin (2011) Cancers 3:3856). Numerous animal tumormodel studies have demonstrated that cytokines have broad anti-tumoractivity and this has been translated into a number of cytokine-basedapproaches for cancer therapy (Lee and Margoli, supra). Recent yearshave seen a number of cytokines, including GM-CSF, IL-7, IL-12, IL-15,IL-18 and IL-21, enter clinical trials for patients with advanced cancer(Lee and Margoli, supra).

Examples of cytokines that may be used in the ADCs include, but are notlimited to, parathyroid hormone; thyroxine; insulin; proinsulin;relaxin; prorelaxin; glycoprotein hormones such as follicle stimulatinghormone (FSH), thyroid stimulating hormone (TSH), and luteinizinghormone (LH); hepatic growth factor; fibroblast growth factor;prolactin; placental lactogen; tumor necrosis factor;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF; platelet-growthfactor; transforming growth factors (TGFs); insulin-like growth factor-Iand -II; erythropoietin (EPO); osteoinductive factors; interferons suchas interferon α, β, and γ, colony stimulating factors (CSFs);granulocyte-macrophage-C-SF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; tumor necrosis factor; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines. Thus, in one embodiment, the disclosureprovides an ADC comprising an anti-LY6H antibody described herein and acytokine.

The anti-LY6H antibodies may be conjugated to at least one colonystimulating factor (CSF). Colony stimulating factors (CSFs) are growthfactors that assist the bone marrow in making red blood cells. Becausesome cancer treatments (e.g., chemotherapy) can affect white blood cells(which help fight infection), colony-stimulating factors may beintroduced to help support white blood cell levels and strengthen theimmune system. Colony-stimulating factors may also be used following abone marrow transplant to help the new marrow start producing whiteblood cells. Representative examples of CSFs that may be used in theanti-LY6H ADCs include, but are not limited to erythropoietin (Epoetin),filgrastim (Neopogen (also known as granulocyte colony-stimulatingfactor (G-CSF); Amgen, Inc.), sargramostim (leukine(granulocyte-macrophage colony-stimulating factor and GM-CSF); GenzymeCorporation), promegapoietin, and Oprelvekin (recombinant IL-11; Pfizer,Inc.). Thus, in one embodiment, an ADC may comprise an anti-LY6Hantibody described herein and a CSF.

4. Alkylating Agents

The anti-LY6H antibodies may be conjugated to one or more alkylatingagent(s). Alkylating agents are a class of antineoplastic compounds thatattaches an alkyl group to DNA. Examples of alkylating agents that maybe used in the ADCs include, but are not limited to, alkyl sulfonates,ethylenimimes, methylamine derivatives, epoxides, nitrogen mustards,nitrosoureas, triazines and hydrazines.

5. DNA Damaging Agents

In one embodiment, the antibodies and antigen-binding portions thereofdescribed herein may be conjugated to one or more DNA damaging agents.The term “DNA damaging agents”, as used herein, refers to any chemicalcompound or treatment method that induces DNA damage. Examples of DNAdamaging agents that may be used in the ADCs include, but are notlimited to, actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin,hexamethylmelamineoxaliplatin, iphosphamide, melphalan,merchlorethamine, mitomycin, mitoxantrone, nemorubicin (MMDX),nitrosourea, plicamycin, procarbazine, PNU-159682(3′-deamino-3″,4′-anhydro-[2″(S)-methoxy-3″(R)-hydroxy-4″-morpholinyl]doxorubicin), taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16).

In one embodiment, anti-LY6H antibodies are conjugated to PNU-159682.PNU-159682 is a bioactivation product of nemorubicin (MMDX), and has apotency in vitro that is more than 1000 times that of the nemorubicindrug and shows high antitumor activity in vivo (Sabatino, M. A., et al.Mol Cancer. 9: 259. 2010). In one embodiment, the anti-LY6H antibodiesare conjugated to PNU-159682 via a linker. In one embodiment, the linkerconjugating the anti-LY6H antibody to PNU-159682 is a cleavable linker.In yet another embodiment, the linker conjugating the anti-LY6H antibodyto PNU-159682 is a non-cleavable linker In one embodiment, the antibodyis coupled to a single drug and, therefore, has a DAR of 1. In certainembodiments, the ADC will have a DAR of 2 to 8, 2 to 6, or 2 to 4.

DNA damaging agents include DNA alkylating agents. DNA alkylating agentsare a class of antineoplastic compounds that attaches an alkyl group(C_(n)H_(2n+1)) to DNA at a guanine base of DNA. Examples of DNAalkylating agents that may be used in the ADCs include, but are notlimited to, alkyl sulfonates (e.g., busulfan), ethylenimimes (e.g.,altretamine and thiotepa), methylamine derivatives, epoxides, nitrogenmustards (e.g., bendamustine, chlorambucil, cyclophosphamide,ifosfamide, mechlorethamine, melphalan), nitrosoureas (e.g., carmustine,lomustine, and streptozocin), triazines (e.g., dacarbazine andtemozolomide), and hydrazines.

DNA damaging agents also include indolino-benzodiazepines (IGNs). IGNsrepresent a chemical class of cytotoxic molecules with high in vitropotency (IC₅₀ values in the low pmol/L range) toward cancer cells.Examples of IGN DNA alkylating agents that can be used as a cytotoxicpayload in an ADC are described in Miller et al. (2016) Molecular CancerTherapeutics, 15(8)). The IGN compounds described in Miller et al. bindto the minor groove of DNA followed by covalent reaction of guanineresidues with the two imine functionalities in the molecule resulting incrosslinking of DNA. The structure of an exemplary IGN is providedbelow.

In one embodiment, a DNA damaging agent may also include apyrrolobenzodiazepine (PBD) or pyridinobenzodiazepine (PDD) (see, e.g.,N. Veillard et al. “Pyridinobenzodiazepines (PDDs): A new class ofsequence-selective DNA mono-alkylating ADC payloads with lowhydrophobicity” [abstract]. In: Proceedings of the 109th Annual Meetingof the American Association for Cancer Research; 2018 Apr. 14-18;Chicago, Ill. Philadelphia (Pa.): AACR; 2018. Abstract no 736/3 andStefano J. E., et al. (2013) Micro- and Mid-Scale Maleimide-BasedConjugation of Cytotoxic Drugs to Antibody Hinge Region Thiols for TumorTargeting. In: Ducry L. (eds) Antibody-Drug Conjugates. Methods inMolecular Biology (Methods and Protocols), vol 1045. Humana Press,Totowa, N.J.)).

In another embodiment, the DNA damaging agent is a PARP inhibitor, e.g.,olaparib, rucaparib, niraparib, or iniparib. In one embodiment, the PARPinhibitor is olaparib. In one embodiment, the PARP inhibitor isrucaparib. In one embodiment, the PARP inhibitor is niraparib. In oneembodiment, the PARP inhibitor is iniparib. In one embodiment, the agentis a saporin toxin.

6. Antiangiogenic Agents

In one aspect, the anti-LY6H antibodies described herein are conjugatedto at least one antiangiogenic agent. Antiangiogenic agents inhibit thegrowth of new blood vessels. Antiangiogenic agents exert their effectsin a variety of ways. In some embodiments, these agents interfere withthe ability of a growth factor to reach its target. For example,vascular endothelial growth factor (VEGF) is one of the primary proteinsinvolved in initiating angiogenesis by binding to particular receptorson a cell surface. Thus, certain antiangiogenic agents, that prevent theinteraction of VEGF with its cognate receptor, prevent VEGF frominitiating angiogenesis. In other embodiments, these agents interferewith intracellular signaling cascades. For example, once a particularreceptor on a cell surface has been triggered, a cascade of otherchemical signals is initiated to promote the growth of blood vessels.Thus, certain enzymes, for example, some tyrosine kinases, that areknown to facilitate intracellular signaling cascades that contribute to,for example, cell proliferation, are targets for cancer treatment. Inother embodiments, these agents interfere with intercellular signalingcascades. Yet, in other embodiments, these agents disable specifictargets that activate and promote cell growth or by directly interferingwith the growth of blood vessel cells. Angiogenesis inhibitoryproperties have been discovered in more than 300 substances withnumerous direct and indirect inhibitory effects.

Representative examples of antiangiogenic agents that may be used in theADCs include, but are not limited to, angiostatin, ABX EGF, C1-1033,PKI-166, EGF vaccine, EKB-569, GW2016, ICR-62, EMD 55900, CP358,PD153035, AG1478, IMC-C225 (Erbitux, ZD1839 (Iressa), OSI-774, Erlotinib(tarceva), angiostatin, arrestin, endostatin, BAY 12-9566 andw/fluorouracil or doxorubicin, canstatin, carboxyamidotriozole and withpaclitaxel, EMD121974, S-24, vitaxin, dimethylxanthenone acetic acid,IM862, Interleukin-12, Interleukin-2, NM-3, HuMV833, PTK787, RhuMab,angiozyme (ribozyme), IMC-1C11, Neovastat, marimstat, prinomastat,BMS-275291, COL-3, MM1270, SU101, SU6668, SU11248, SU5416, withpaclitaxel, with gemcitabine and cisplatin, and with irinotecan andcisplatin and with radiation, tecogalan, temozolomide and PEG interferonα1b, tetrathiomolybdate, TNP-470, thalidomide, CC-5013 and withtaxotere, tumstatin, 2-methoxyestradiol, VEGF trap, mTOR inhibitors(deforolimus, everolimus (Afinitor, Novartis PharmaceuticalCorporation), and temsirolimus (Torisel, Pfizer, Inc.)), tyrosine kinaseinhibitors (e.g., erlotinib (Tarceva, Genentech, Inc.), imatinib(Gleevec, Novartis Pharmaceutical Corporation), gefitinib (Iressa,AstraZeneca Pharmaceuticals), dasatinib (Sprycel, Brystol-Myers Squibb),sunitinib (Sutent, Pfizer, Inc.), nilotinib (Tasigna, NovartisPharmaceutical Corporation), lapatinib (Tykerb, GlaxoSmithKlinePharmaceuticals), sorafenib (Nexavar, Bayer and Onyx), phosphoinositide3-kinases (PI3K).

7. Antimetabolites

The anti-LY6H antibodies may be conjugated to at least oneantimetabolite. Antimetabolites are types of chemotherapy treatmentsthat are very similar to normal substances within the cell. When thecells incorporate an antimetabolite into the cellular metabolism, theresult is negative for the cell, e.g., the cell is unable to divide.Antimetabolites are classified according to the substances with whichthey interfere. Examples of antimetabolies that may be used in the ADCsinclude, but are not limited to, a folic acid antagonist (e.g.,methotrexate), a pyrimidine antagonist (e.g., 5-Fluorouracil,Foxuridine, Cytarabine, Capecitabine, and Gemcitabine), a purineantagonist (e.g., 6-Mercaptopurine and 6-Thioguanine) and an adenosinedeaminase inhibitor (e.g., Cladribine, Fludarabine, Nelarabine andPentostatin), as described in more detail below.

8. Boron-Containing Agents

The anti-LY6H antibody may be conjugated to at least one boroncontaining agent. Boron-containing agents comprise a class of cancertherapeutic compounds which interfere with cell proliferation.Representative examples of boron containing agents include, but are notlimited, to borophycin and bortezomib (Velcade, MilleniumPharmaceuticals).

9. Chemoprotective Agents

The anti-LY6H antibodies may be conjugated to at least onechemoprotective agent. Chemoprotective drugs are a class of compounds,which help protect the body against specific toxic effects ofchemotherapy. Chemoprotective agents may be administered with variouschemotherapies in order to protect healthy cells from the toxic effectsof chemotherapy drugs, while simultaneously allowing the cancer cells tobe treated with the administered chemotherapeutic. Representativechemoprotective agents include, but are not limited to amifostine(Ethyol, Medimmune, Inc.), which is used to reduce renal toxicityassociated with cumulative doses of cisplatin, dexrazoxane (Totect,Apricus Pharma; Zinecard), for the treatment of extravasation caused bythe administration of anthracycline (Totect), and for the treatment ofcardiac-related complications caused by the administration of theantitumor antibiotic doxorubicin (Zinecard), and mesna (Mesnex,Bristol-Myers Squibb), which is used to prevent hemorrhagic cystitisduring chemotherapy treatment with ifocfamide.

10. Photoactive Therapeutic Agents

The anti-LY6H antibodies may be conjugated to at least one photoactivetherapeutic agent. Photoactive therapeutic agents include compounds thatcan be deployed to kill treated cells upon exposure to electromagneticradiation of a particular wavelength. Therapeutically relevant compoundsabsorb electromagnetic radiation at wavelengths which penetrate tissue.In preferred embodiments, the compound is administered in a non-toxicform that is capable of producing a photochemical effect that is toxicto cells or tissue upon sufficient activation. In other preferredembodiments, these compounds are retained by cancerous tissue and arereadily cleared from normal tissues. Non-limiting examples includevarious chromagens and dyes.

11. Radionuclide Agents (Radioactive Isotopes)

The anti-LY6H antibodies may be conjugated to at least one radionuclideagent. Radionuclide agents comprise agents that are characterized by anunstable nucleus that is capable of undergoing radioactive decay. Thebasis for successful radionuclide treatment depends on sufficientconcentration and prolonged retention of the radionuclide by the cancercell. Other factors to consider include the radionuclide half-life, theenergy of the emitted particles, and the maximum range that the emittedparticle can travel. In preferred embodiments, the therapeutic agent isa radionuclide selected from the group consisting of ¹¹¹In, ¹⁷⁷Lu,²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc,¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re,²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr,¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb. Also preferred areradionuclides that substantially decay with Auger-emitting particles.For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111 1,Sb-119, 1-125, Ho-161, Os-189m and Ir-192. Decay energies of usefulbeta-particle-emitting nuclides are preferably Dy-152, At-211, Bi-212,Ra-223, Rn-219, Po-215, Bi-21 1, Ac-225, Fr-221, At-217, Bi-213 andFm-255. Decay energies of useful alpha-particle-emitting radionuclidesare preferably 2,000-10,000 keV, more preferably 3,000-8,000 keV, andmost preferably 4,000-7,000 keV. Additional potential radioisotopes ofuse include ¹¹C, ¹³N, ¹⁵0, ⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br,^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te,^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr,¹⁶¹Th, ^(!66)Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac,⁷⁶Br, ¹⁶⁹Yb, and the like.

12. Radiosensitizers

The anti-LY6H antibodies may be conjugated to at least oneradiosensitizer. The term “radiosensitizer,” as used herein, is definedas a molecule, preferably a low molecular weight molecule, administeredto animals in therapeutically effective amounts to increase thesensitivity of the cells to be radiosensitized to electromagneticradiation and/or to promote the treatment of diseases that are treatablewith electromagnetic radiation. Radiosensitizers are agents that makecancer cells more sensitive to radiation therapy, while typically havingmuch less of an effect on normal cells. Thus, the radiosensitizer can beused in combination with a radiolabeled antibody or ADC. The addition ofthe radiosensitizer can result in enhanced efficacy when compared totreatment with the radiolabeled antibody or antibody fragment alone.Radiosensitizers are described in D. M. Goldberg (ed.), Cancer Therapywith Radiolabeled Antibodies, CRC Press (1995). Examples ofradiosensitizers include gemcitabine, 5-fluorouracil, taxane, andcisplatin.

Radiosensitizers may be activated by the electromagnetic radiation ofX-rays. Representative examples of X-ray activated radiosensitizersinclude, but are not limited to, the following: metronidazole,misonidazole, desmethylmisonidazole, pimonidazole, etanidazole,nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145, nicotinamide,5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR),bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin,and therapeutically effective analogs and derivatives of the same.Alternatively, radiosensitizers may be activated using photodynamictherapy (PDT). Representative examples of photodynamic radiosensitizersinclude, but are not limited to, hematoporphyrin derivatives,Photofrin(r), benzoporphyrin derivatives, NPe6, tin etioporphyrin(SnET2), pheoborbide a, bacteriochlorophyll a, naphthalocyanines,phthalocyanines, zinc phthalocyanine, and therapeutically effectiveanalogs and derivatives of the same.

13. Topoisomerase Inhibitors

The anti-LY6H antibodies may be conjugated to at least one topoisomeraseinhibitor. Topoisomerase inhibitors are chemotherapy agents designed tointerfere with the action of topoisomerase enzymes (topoisomerase I andII), which are enzymes that control the changes in DNA structure bycatalyzing then breaking and rejoining of the phosphodiester backbone ofDNA strands during the normal cell cycle. Representative examples of DNAtopoisomerase I inhibitors include, but are not limited to,camptothecins and its derivatives irinotecan (CPT-11, Camptosar, Pfizer,Inc.) and topotecan (Hycamtin, GlaxoSmithKline Pharmaceuticals).Representative examples of DNA topoisomerase II inhibitors include, butare not limited to, amsacrine, daunorubicin, doxotrubicin,epipodophyllotoxins, ellipticines, epirubicin, etoposide, razoxane, andteniposide.

14. Tyrosine Kinase Inhibitors

The anti-LY6H antibodies may be conjugated to at least one tyrosinekinase inhibitor. Tyrosine kinases are enzymes within the cell thatfunction to attach phosphate groups to the amino acid tyrosine. Byblocking the ability of protein tyrosine kinases to function, tumorgrowth may be inhibited. Examples of tyrosine kinases that may be usedon the ADCs include, but are not limited to, Axitinib, Bosutinib,Cediranib, Dasatinib, Erlotinib, Gefitinib, Imatinib, Lapatinib,Lestaurtinib, Nilotinib, Semaxanib, Sunitinib, and Vandetanib.

15. Other Agents

Examples of other agents that may be used in the ADCs include, but arenot limited to, abrin (e.g. abrin A chain), alpha toxin, Aleuritesfordii proteins, amatoxin, crotin, curcin, dianthin proteins, diptheriatoxin (e.g. diphtheria A chain and nonbinding active fragments ofdiphtheria toxin), deoxyribonuclease (Dnase), gelonin, mitogellin,modeccin A chain, Momordica charantia inhibitor, neomycin, onconase,phenomycin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),pokeweed antiviral protein, Pseudomonas endotoxin, Pseudomonas exotoxin(e.g. exotoxin A chain (from Pseudomonas aeruginosa)), restrictocin,ricin A chain, ribonuclease (Rnase), Sapaonaria officinalis inhibitor,saporin, alpha-sarcin, Staphylcoccal enterotoxin-A, tetanus toxin,cisplatin, carboplatin, and oxaliplatin (Eloxatin, Sanofi Aventis),proteasome inhibitors (e.g. PS-341 [bortezomib or Velcade]), HDACinhibitors (vorinostat (Zolinza, Merck & Company, Inc.)), belinostat,entinostat, mocetinostat, and panobinostat), COX-2 inhibitors,substituted ureas, heat shock protein inhibitors (e.g. Geldanamycin andits numerous analogs), adrenocortical suppressants, and thetricothecenes. (See, for example, WO 93/21232). Other agents alsoinclude asparaginase (Espar, Lundbeck Inc.), hydroxyurea, levamisole,mitotane (Lysodren, Bristol-Myers Squibb), and tretinoin (Renova,Valeant Pharmaceuticals Inc.).

It should be noted that the aforementioned groups of drug moieties thatmay be used in the anti-LY6H ADCs are not exclusive, in that certainexamples of drugs may be found in more than one category, e.g.,ansamitocins are both mitotic inhibitors and antitumor antibiotics.

All stereoisomers of the above drug moieties are contemplated for useherein, i.e. any combination of R and S configurations at the chiralcarbons of D.

The above agents (i.e., naked agents not conjugated to an antibody) mayalso be used in combination therapies with the anti-LY6H antibodiesdescribed herein. In one embodiment, anti-LY6H antibodies or ADCs areused with any of the foregoing agents in a combination therapy to treatcancer, where the agent is administered prior to, at the same time as,or following administration of the anti-LY6H antibody or ADC to thesubject.

B. Anti-LY6H ADCs: Exemplary Linkers

An anti-LY6H ADC comprises an anti-LY6H antibody and at least onedrug(s), whereby the antibody and the at least one drug are conjugatedby a linker. The term “linker,” as used herein, refers to a chemicalmoiety that may be bifunctional or multifunctional, and is used toattach an antibody to a drug moiety. A linker may include oneconjugating component or may include multiple components.

For example, the linker may include a spacer, which is a moiety thatextends the drug linkage to avoid, for example, shielding the activesite of the antibody or improving the solubility of the ADC. Otherexamples of components of linkers include a stretcher unit and an aminoacid unit.

Two methods are commonly used for conjugating drugs to antibodies:alkylation of reduced interchain cysteine disulfides through anenzymatically non-cleavable maleimido or simple and cleavable disulfidelinker, and acylation of lysines by cleavable linear amino acids.

In one aspect, a linker covalently attaches an antibody to a drugmoiety. An ADC is prepared using a linker having reactive functionalityfor binding to the antibody and the drug. For example, a cysteine thiol,or an amine, e.g., N-terminus or amino acid side chain such as lysine,of the antibody may form a bond with a functional group of the linker.

In one aspect, a linker has a functionality that is capable of reactingwith a free cysteine present on an antibody to form a covalent bond.Nonlimiting exemplary such reactive functionalities include maleimide,haloacetamides, α-haloacetyl, activated esters such as succinimideesters, 4-nitrophenyl esters, pentafluorophenyl esters,tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates, and isothiocyanates. See, e.g., the conjugationmethod at page 766 of Klussman, et al (2004), Bioconjugate Chemistry15(4):765-773.

In some embodiments, a linker has a functionality that is capable ofreacting with an electrophilic group present on an antibody. Exemplarysuch electrophilic groups include, but are not limited to, aldehyde andketone carbonyl groups. In some embodiments, a heteroatom of thereactive functionality of the linker can react with an electrophilicgroup on an antibody and form a covalent bond to an antibody unit.Nonlimiting exemplary such reactive functionalities include, but are notlimited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide.

Suitable linkers include, for example, cleavable and non-cleavablelinkers. A linker may be a “cleavable linker,” facilitating release of adrug. Nonlimiting exemplary cleavable linkers include acid-labilelinkers (e.g., comprising hydrazone), protease-sensitive (e.g.,peptidase-sensitive) linkers, photolabile linkers, ordisulfide-containing linkers (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020). A cleavable linker is typicallysusceptible to cleavage under intracellular conditions. Suitablecleavable linkers include, for example, a peptide linker cleavable by anintracellular protease, such as lysosomal protease or an endosomalprotease. In exemplary embodiments, the linker can be a dipeptidelinker, such as a valine-citrulline (val-cit) or a phenylalanine-lysine(phe-lys) linker.

Linkers are preferably stable extracellularly in a sufficient manner tobe therapeutically effective. Before transport or delivery into a cell,the ADC is preferably stable and remains intact, i.e. the antibodyremains conjugated to the drug moiety. Linkers that are stable outsidethe target cell may be cleaved at some efficacious rate once inside thecell. Thus, an effective linker will: (i) maintain the specific bindingproperties of the antibody; (ii) allow delivery, e.g., intracellulardelivery, of the drug moiety; and (iii) maintain the therapeutic effect,e.g., cytotoxic effect, of a drug moiety.

In one embodiment, the linker is cleavable under intracellularconditions, such that cleavage of the linker sufficiently releases thedrug from the antibody in the intracellular environment to betherapeutically effective. In some embodiments, the cleavable linker ispH-sensitive, i.e., sensitive to hydrolysis at certain pH values.Typically, the pH-sensitive linker is hydrolyzable under acidicconditions. For example, an acid-labile linker that is hydrolyzable inthe lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone,cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used.(See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchikand Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989,Biol. Chem. 264:14653-14661.) Such linkers are relatively stable underneutral pH conditions, such as those in the blood, but are unstable atbelow pH 5.5 or 5.0, the approximate pH of the lysosome. In certainembodiments, the hydrolyzable linker is a thioether linker (such as,e.g., a thioether attached to the therapeutic agent via an acylhydrazonebond (see, e.g., U.S. Pat. No. 5,622,929).

In other embodiments, the linker is cleavable under reducing conditions(e.g., a disulfide linker) A variety of disulfide linkers are known inthe art, including, for example, those that can be formed using SATA(N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935).

In some embodiments, the linker is cleavable by a cleaving agent, e.g.,an enzyme, that is present in the intracellular environment (e.g.,within a lysosome or endosome or caveolea). The linker can be, e.g., apeptidyl linker that is cleaved by an intracellular peptidase orprotease enzyme, including, but not limited to, a lysosomal or endosomalprotease. In some embodiments, the peptidyl linker is at least two aminoacids long or at least three amino acids long. Cleaving agents caninclude cathepsins B and D and plasmin, all of which are known tohydrolyze dipeptide drug derivatives resulting in the release of activedrug inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm.Therapeutics 83:67-123). Most typical are peptidyl linkers that arecleavable by enzymes that are present in LY6H-expressing cells. Examplesof such linkers are described, e.g., in U.S. Pat. No. 6,214,345,incorporated herein by reference in its entirety and for all purposes.In a specific embodiment, the peptidyl linker cleavable by anintracellular protease is a Val-Cit linker or a Phe-Lys linker (see,e.g., U.S. Pat. No. 6,214,345, which describes the synthesis ofdoxorubicin with the val-cit linker) One advantage of usingintracellular proteolytic release of the therapeutic agent is that theagent is typically attenuated when conjugated and the serum stabilitiesof the conjugates are typically high.

In other embodiments, the linker is a malonate linker (Johnson et al.,1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau etal., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3′-N-amide analog(Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released, for example, by antibody degradation. See U.S. PublicationNo. 20050238649 incorporated by reference herein in its entirety. An ADCcomprising a non-cleavable linker may be designed such that the ADCremains substantially outside the cell and interacts with certainreceptors on a target cell surface such that the binding of the ADCinitiates (or prevents) a particular cellular signaling pathway.

In some embodiments, the linker is substantially hydrophilic linker(e.g., PEG4Mal and sulfo-SPDB). A hydrophilic linker may be used toreduce the extent to which the drug may be pumped out of resistantcancer cells through MDR (multiple drug resistance) or functionallysimilar transporters.

In other embodiments, upon cleavage, the linker functions to directly orindirectly inhibit cell growth and/or cell proliferation. For example,in some embodiments, the linker, upon cleavage, can function as anintercalating agent, thereby inhibiting macromolecular biosynthesis(e.g. DNA replication, RNA transcription, and/or protein synthesis).

In other embodiments, the linker is designed to facilitate bystanderkilling (the killing of neighboring cells) through diffusion of thelinker-drug and/or the drug alone to neighboring cells. In other,embodiments, the linker promotes cellular internalization.

The presence of a sterically hindered disulfide can increase thestability of a particular disulfide bond, enhancing the potency of theADC. Thus, in one embodiment, the linker includes a sterically hindereddisulfide linkage. A sterically hindered disulfide refers to a disulfidebond present within a particular molecular environment, wherein theenvironment is characterized by a particular spatial arrangement ororientation of atoms, typically within the same molecule or compound,which prevents or at least partially inhibits the reduction of thedisulfide bond. Thus, the presence of bulky (or sterically hindering)chemical moieties and/or bulky amino acid side chains proximal to thedisulfide bond prevents or at least partially inhibits the disulfidebond from potential interactions that would result in the reduction ofthe disulfide bond.

Notably, the aforementioned linker types are not mutually exclusive. Forexample, in one embodiment, the linker used in the anti-LY6H ADCsdescribed herein is a non-cleavable linker that promotes cellularinternalization.

In some embodiments, the ADC has the following formula (formula I):

Ab-(L-D)ntm (I)

or a pharmaceutically acceptable salt or solvate thereof; wherein Ab isthe antibody, e.g., anti-LY6H antibody, and (L-D) is a Linker-Drugmoiety. The Linker-Drug moiety is made of L- which is a Linker, and -D,which is a drug moiety having, for example, cytostatic, cytotoxic, orotherwise therapeutic activity against a target cell, e.g., a cellexpressing LY6H; and n is an integer from 1 to 20.

In some embodiments, n ranges from 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to4, 1 to 3, 1 to 2, or is 1.

In some embodiments, the -D moieties are the same. In yet anotherembodiment, the -D moieties are different.

In some embodiments, a linker component comprises an “amino acid unit.”In some such embodiments, the amino acid unit allows for cleavage of thelinker by a protease, thereby facilitating release of the drug from theimmunoconjugate upon exposure to intracellular proteases, such aslysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784).Exemplary amino acid units include, but are not limited to, dipeptides,tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptidesinclude, but are not limited to, valine-citrulline (vc or val-cit),alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk orphe-lys); phenylalanine-homolysine (phe-homolys); andN-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include,but are not limited to, glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally and/or minor amino acids and/ornon-naturally occurring amino acid analogs, such as citrulline Aminoacid units can be designed and optimized for enzymatic cleavage by aparticular enzyme, for example, a tumor-associated protease, cathepsinB, C and D, or a plasmin protease.

In one embodiment, the amino acid unit is valine-citrulline (vc orval-cit). In another aspect, the amino acid unit is phenylalanine-lysine(i.e., fk). In yet another aspect of the amino acid unit, the amino acidunit is N-methylvaline-citrulline. In yet another aspect, the amino acidunit is 5-aminovaleric acid, homo phenylalanine lysine,tetraisoquinolinecarboxylate lysine, cyclohexylalanine lysine,isonepecotic acid lysine, beta-alanine lysine, glycine serine valineglutamine and isonepecotic acid.

Another approach for the generation of ADCs involves the use ofheterobifunctional cross-linkers which link the anti-LY6H antibody tothe drug moiety. Examples of cross-linkers that may be used includeN-succinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate or the highlywater-soluble analog N-sulfosuccinimidyl4-(5-nitro-2-pyridyldithio)-pentanoate,N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB),N-succinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SNPB), andN-sulfosuccinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SSNPB),N-succinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)pentanoate (SMNP),N-succinimidyl-4-(5-N,N-dimethylcarboxamido-2-pyridyldithio) butyrate(SCPB) orN-sulfosuccinimidyl4-(5-N,N-dimethylcarboxamido-2-pyridyldithio)butyrate (SSCPB)). The antibodies may be modified with the cross-linkersN-succinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate,N-sulfosuccinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate, SPDB, SNPB,SSNPB, SMNP, SCPB, or SSCPB can then react with a small excess of aparticular drug that contains a thiol moiety to give excellent yields ofan ADC (see also U.S. Pat. No. 6,913,748, incorporated by referenceherein).

In one embodiment, charged linkers (also referred to as pro-chargedlinkers) are used to conjugate anti-LY6H antibodies to drugs to formADCs. Charged linkers include linkers that become charged after cellprocessing. The presence of a charged group(s) in the linker of aparticular ADC or on the drug after cellular processing provides severaladvantages, such as (i) greater water solubility of the ADC, (ii)ability to operate at a higher concentration in aqueous solutions, (iii)ability to link a greater number of drug molecules per antibody,potentially resulting in higher potency, (iv) potential for the chargedconjugate species to be retained inside the target cell, resulting inhigher potency, and (v) improved sensitivity of multidrug resistantcells, which would be unable to export the charged drug species from thecell. Examples of some suitable charged or pro-charged cross-linkers andtheir synthesis are shown in FIGS. 1 to 10 of U.S. Pat. No. 8,236,319,and are incorporated by reference herein. Preferably, the charged orpro-charged cross-linkers are those containing sulfonate, phosphate,carboxyl or quaternary amine substituents that significantly increasethe solubility of the ADCs, especially for ADCs with 2 to 20 conjugateddrugs. Conjugates prepared from linkers containing a pro-charged moietywould produce one or more charged moieties after the conjugate ismetabolized in a cell.

Additional examples of linkers that can be used with the compositionsand methods include valine-citrulline; maleimidocaproyl; amino benzoicacids; p-aminobenzylcarbamoyl (PAB); lysosomal enzyme-cleavable linkers;maleimidocaproyl-polyethylene glycol (MC(PEG)6-OH); N-methyl-valinecitrulline; N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC);

N-Succinimidyl 4-(2-pyridyldithio)butanoate (SPDB); and N-Succinimidyl4-(2-pyridylthio)pentanoate (SPP) (See also US 2011/0076232). Anotherlinker for use includes an avidin-biotin linkage to provide anavidin-biotin-containing ADC (See also U.S. Pat. No. 4,676,980, PCTpublication Nos. WO1992/022332A2, WO1994/016729A1, WO1995/015770A1,WO1997/031655A2, WO1998/035704A1, WO1999/019500A1, WO2001/09785A2,WO2001/090198A1, WO2003/093793A2, WO2004/050016A2, WO2005/081898A2,WO2006/083562A2, WO2006/089668A1, WO2007/150020A1, WO2008/135237A1,WO2010/111198A1, WO2011/057216A1, WO2011/058321A1, WO2012/027494A1, andEP77671B1), wherein some such linkers are resistant to biotinidasecleavage. Additional linkers that may be used include a cohesin/dockerinpair to provide a cohesion-dockerin-containing ADC (See PCT publicationNos. WO2008/097866A2, WO2008/097870A2, WO2008/103947A2, andWO2008/103953A2).

Additional linkers may contain non-peptide polymers (examples include,but are not limited to, polyethylene glycol, polypropylene glycol,polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran,polyvinyl ethyl ether, PLA (poly(lactic acid)), PLGA (poly(lacticacid-glycolic acid)), and combinations thereof, wherein a preferredpolymer is polyethylene glycol) (See also PCT publication No.WO2011/000370). Additional linkers are also described in WO 2004-010957,U.S. Publication No. 20060074008, U.S. Publication No. 20050238649, andU.S. Publication No. 20060024317, each of which is incorporated byreference herein in its entirety).

For an ADC comprising a maytansinoid, many positions on maytansinoidscan serve as the position to chemically link the linking moiety. In oneembodiment, maytansinoids comprise a linking moiety that contains areactive chemical group are C-3 esters of maytansinol and its analogswhere the linking moiety contains a disulfide bond and the chemicalreactive group comprises a N-succinimidyl or N-sulfosuccinimidyl ester.For example, the C-3 position having a hydroxyl group, the C-14 positionmodified with hydroxymethyl, the C-15 position modified with hydroxy andthe C-20 position having a hydroxy group are all useful. The linkingmoiety most preferably is linked to the C-3 position of maytansinol.

The conjugation of the drug to the antibody via a linker can beaccomplished by any technique known in the art. A number of differentreactions are available for covalent attachment of drugs and linkers toantibodies. This may be accomplished by reaction of the amino acidresidues of the antibody, including the amine groups of lysine, the freecarboxylic acid groups of glutamic and aspartic acid, the sulfhydrylgroups of cysteine and the various moieties of the aromatic amino acids.One of the most commonly used non-specific methods of covalentattachment is the carbodiimide reaction to link a carboxy (or amino)group of a compound to amino (or carboxy) groups of the antibody.Additionally, bifunctional agents such as dialdehydes or imidoestershave been used to link the amino group of a compound to amino groups ofan antibody. Also available for attachment of drugs to antibodies is theSchiff base reaction. This method involves the periodate oxidation of adrug that contains glycol or hydroxy groups, thus forming an aldehydewhich is then reacted with the binding agent. Attachment occurs viaformation of a Schiff base with amino groups of the antibody.Isothiocyanates can also be used as coupling agents for covalentlyattaching drugs to antibodies. Other techniques are known to the skilledartisan and within the scope of the disclosure.

In certain embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. Incertain embodiments, reactive groups are used on the drug or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with theanti-LY6H antibody under appropriate conditions. The synthesis andstructure of exemplary linkers, stretcher units, amino acid units,self-immolative spacer units are described in U.S. Patent ApplicationPublication Nos. 20030083263, 20050238649 and 20050009751, each if whichis incorporated herein by reference.

Stability of the ADC may be measured by standard analytical techniquessuch as mass spectroscopy, HPLC, and the separation/analysis techniqueLC/MS.

IV. Uses of Anti-LY6H Antibodies and Anti-LY6H ADCs

The antibodies and antibody portions (and ADCs) preferably are capableof neutralizing LY6H activity, in particular human LY6H activity, bothin vivo and in vitro. Accordingly, such antibodies and antibody portionscan be used to inhibit hLY6H activity, e.g., in a cell culturecontaining hLY6H, in human subjects or in other mammalian subjectshaving LY6H with which an antibody disclosed herein cross-reacts. In oneembodiment, the disclosure provides a method for inhibiting hLY6Hactivity comprising contacting hLY6H with an antibody or antibodyportion such that hLY6H activity is inhibited. For example, in a cellculture containing, or suspected of containing hLY6H, an antibody orantibody portion can be added to the culture medium to inhibit hLY6Hactivity in the culture.

In another embodiment, disclosed herein is a method for reducing hLY6Hactivity in a subject, advantageously from a subject suffering from aLY6H associated disorder, e.g., cancer, including, but not limited to,small cell lung cancer (SCLC), e.g., classic SCLC or variant SCLC,gastrointestinal stromal tumor (GIST), T cell acute lymphoblasticleukemia (T-ALL), glioblastoma and soft tissue sarcoma, or a disorder inwhich LY6H activity is detrimental. The disclosure provides methods forreducing LY6H activity in a subject suffering from such a disease ordisorder, which method comprises administering to the subject anantibody or antibody portion of the disclosure such that LY6H activityin the subject is reduced. Preferably, the LY6H is human LY6H, and thesubject is a human subject. Alternatively, the subject can be a mammalexpressing a LY6H to which antibodies of the disclosure are capable ofbinding. Still further the subject can be a mammal into which LY6H hasbeen introduced (e.g., by administration of LY6H or by expression of aLY6H transgene). Antibodies of the disclosure can be administered to ahuman subject for therapeutic purposes. Moreover, antibodies of thedisclosure can be administered to a non-human mammal expressing a LY6Hwith which the antibody is capable of binding for veterinary purposes oras an animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies of the disclosure (e.g., testing of dosages and time coursesof administration).

As used herein, the term “a disorder in which LY6H activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of LY6H in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which LY6Hactivity is detrimental is a disorder in which reduction of LY6Hactivity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of LY6H in a biological cell, fluid or tissue of asubject suffering from the disorder (e.g., an increase in theconcentration of LY6H in a tumor, serum, plasma, synovial fluid, etc. ofthe subject), which can be detected, for example, using an anti-LY6Hantibody as described above.

Non-limiting examples of disorders that can be treated with theantibodies, or antigen binding fragments thereof, include thosedisorders discussed below. For example, suitable disorders include, butare not limited to, a variety of cancers including, but not limited to,small cell lung cancer (SCLC), e.g., classic SCLC or variant SCLC,gastrointestinal stromal tumor (GIST), T cell acute lymphoblasticleukemia (T-ALL), glioblastoma, soft tissue sarcoma, breast cancer,brain and CNS cancer, head and neck cancer, bladder cancer, renalcancer, ovarian cancer, esophageal cancer, gastric cancer, prostatecancer, uterine cancer, endometrial cancer, pancreatic cancer, livercancer, colorectal cancer, cervical cancer and non small cell lungcancer (NSCLC).

Other examples of cancers that may be treated using the compositions andmethods disclosed herein include, but are not limited to carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include but are not limited tobreast cancer (Luminal A, TNBC, Ductal), prostate cancer, squamous celltumors, squamous cell carcinoma (e.g., squamous cell lung cancer orsquamous cell head and neck cancer), neuroendocrine tumors, urothelialcancer, vulvar cancer, mesothelioma, liver cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, lung cancer,small cell lung cancer, non-small cell lung cancer, cutaneous orintraocular malignant melanoma, renal cancer, uterine cancer, ovariancancer, colorectal cancer, colon cancer, rectal cancer, cancer of theanal region, stomach cancer, testicular cancer, uterine cancer,carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the cervix, carcinoma of the vagina, carcinoma of thevulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of thesmall intestine, cancer of the endocrine system, cancer of theparathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue,cancer of the urethra, cancer of the penis, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, environmentally induced cancers including thoseinduced by asbestos, hematologic malignancies including, for example,multiple myeloma, B-cell lymphoma, Hodgkin lymphoma/primary mediastinalB-cell lymphoma, non-Hodgkin's lymphomas, acute myeloid lymphoma,chronic myelogenous leukemia, chronic lymphoid leukemia, follicularlymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma,immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides,anaplastic large cell lymphoma, T-cell lymphoma, and precursorT-lymphoblastic lymphoma, and any combinations of said cancers. PVNS,acute myeloid leukemia, adrenocortico carcinoma, ladder urothelialcarcinoma, cervical squamous cell carcinoma, endocervicaladenocarcinoma, diffuse large B cell lymphoma, glioblastoma multiforme,chronic lymphocytic leukemia, brain lower grade glioma, head and necksquamous cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma,large squamous cell carcinoma, cutaneous melanoma, ovarial serouscystadenocarcinoma, gastric cancer, soft tissue sarcoma, testicular germcell cancer, thymoma, thyroid carcinoma, uterine corpus endometrialcarcinoma, uterine carcinosarcoma, kidney renal clear cell carcinoma,and kidney renal papillary cell carcinoma. The present invention is alsoapplicable to treatment of metastatic cancers.

In one embodiment, an anti-LY6H ADC of the present invention can be usedto treat a cancer in a subject including, but not limited to Hodgkin'slymphoma, PVNS, acute myeloid leukemia, adrenocortico carcinoma, ladderurothelial carcinoma, breast cancer (Luminal A, TNBC, Ductal), cervicalsquamous cell carcinoma, endocervical adenocarcinoma, colorectaladenocarcinoma, diffuse large B cell lymphoma, non-hodgkin's lymphoma,glioblastoma multiforme, chronic lymphocytic leukemia, brain lower gradeglioma, head and neck squamous cell carcinoma, hepatocellular carcinoma,lung adenocarcinoma, small cell lung cancer, large squamous cellcarcinoma, cutaneous melanoma, ovarial serous cystadenocarcinoma,gastric cancer, soft tissue sarcoma, mesothelioma, pancreaticadenocarcinoma, testicular germ cell cancer, thymoma, thyroid carcinoma,uterine corpus endometrial carcinoma, uterine carcinosarcoma. kidneyrenal clear cell carcinoma, and kidney renal papillary cell carcinoma.

In one embodiment, the antibodies and ADCs disclosed herein are used totreat a solid tumor, e.g., inhibit growth of or decrease size of a solidtumor, overexpressing LY6H or which is LY6H positive. In one embodiment,the antibodies and ADCs disclosed herein are used to treat cancer,including, but not limited to, small cell lung cancer (SCLC),gastrointestinal stromal tumor (GIST), T cell acute lymphoblasticleukemia (T-ALL), glioblastoma, and soft tissue sarcoma. Diseases anddisorders described herein may be treated by anti-LY6H antibodies orADCs, as well as pharmaceutical compositions comprising such anti-LY6Hantibodies or ADCs.

In certain embodiments, the antibodies and ADCs disclosed herein areadministered to a subject in need thereof in order to treat advancedsolid tumor types likely to exhibit elevated levels of LY6H. In certainembodiments, the disclosure includes a method for inhibiting ordecreasing solid tumor growth in a subject having a solid tumor, saidmethod comprising administering an anti-LY6H antibody or ADC describedherein, to the subject having the solid tumor, such that the solid tumorgrowth is inhibited or decreased. In one embodiment, the solid tumor isa small cell lung cancer (SCLC) tumor. In another embodiment, the solidtumor is a gastrointestinal stromal tumor (GIST). In some embodiments,the solid tumor is a glioblastoma tumor. In another embodiment, thesolid tumor is a soft tissue sarcoma tumor. In further embodiments, thesolid tumor is an LY6H expressing solid tumor. In certain embodiments,the disclosure includes a method for treating a cancer that generallydoes not form a solid tumor in a subject, e.g., T cell acutelymphoblastic leukemia (T-ALL), said method comprising administering ananti-LY6H antibody or ADC described herein, to the subject having T cellacute lymphoblastic leukemia (T-ALL), such that T cell acutelymphoblastic leukemia (T-ALL) is inhibited or decreased. In certainembodiments the anti-LY6H antibodies or ADCs described herein areadministered to a subject having a cancer, including, but not limitedto, small cell lung cancer (SCLC), gastrointestinal stromal tumor(GIST), T cell acute lymphoblastic leukemia (T-ALL), glioblastoma, andsoft tissue sarcoma, alone or in combination with an additional agent,e.g., radiation and/or chemotherapy, or an immune checkpoint inhibitor.

In certain embodiments, the disclosure includes a method for inhibitingor decreasing solid tumor growth in a subject having a solid tumor whichwas identified as an LY6H expressing or LY6H positive tumor, said methodcomprising administering an anti-LY6H antibody or ADC described herein,to the subject having the solid tumor, such that the solid tumor growthis inhibited or decreased. In further embodiments, the solid tumor is aprimary tumor.

In certain embodiments, the disclosure includes a method for inhibitingor decreasing solid tumor growth in a subject having a solid tumor whichwas identified as an LY6H expressing tumor, said method comprisingadministering an anti-LY6H antibody or ADC described herein, to thesubject having the solid tumor, such that the solid tumor growth isinhibited or decreased. Methods for identifying LY6H expressing tumorsare known in the art, and include FDA-approved tests and validationassays. For example, these assays may use primers that are specific forthe LY6H gene and/or cDNA and result in the amplification of the LY6Hgene/cDNA, or a portion thereof. The amplified PCR products may besubsequently analyzed, for example, by gel electrophoresis usingstandard methods known in the art to determine the size of the PCRproducts. Such tests may be used to identify tumors that may be treatedwith the methods and compositions described herein.

In another aspect, this application features a method of treating (e.g.,curing, suppressing, ameliorating, delaying or preventing the onset of,or preventing recurrence or relapse of) or preventing a LY6H-associateddisorder, in a subject. The method includes: administering to thesubject a LY6H binding agent (particularly an antagonist), e.g., ananti-LY6H antibody or fragment thereof as described herein, in an amountsufficient to treat or prevent the LY6H-associated disorder. The LY6Hantagonist, e.g., the anti-LY6H antibody or fragment thereof, can beadministered to the subject, alone or in combination with othertherapeutic modalities as described herein.

In some embodiments, the anti-LY6H antibody or fragment thereof used inthe methods of the invention is a human or humanized anti-LY6H antibodyor fragment thereof.

In another embodiment, antibody-dependent cell-mediated cytotoxicity(ADCC) activity is not necessary for anti-LY6H antibodies to inhibittumor growth or reduce tumor size. Accordingly, in one embodiment, anantibody, or antigen binding portion thereof, of the invention comprisesan isotype lacking effector function (e.g., human IgG₄).

Antibodies or ADCs, or antigen binding portions thereof, can be usedalone or in combination to treat such diseases. It should be understoodthat the antibodies or antigen binding portion thereof can be used aloneor in combination with an additional agent, e.g., a therapeutic agent,said additional agent being selected by the skilled artisan for itsintended purpose. For example, the additional agent can be a therapeuticagent art-recognized as being useful to treat the disease or conditionbeing treated by the antibody. The additional agent also can be an agentthat imparts a beneficial attribute to the therapeutic composition,e.g., an agent which affects the viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this disclosure are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limited. The combinations, which arepart of this disclosure, can be the antibodies of the disclosure and atleast one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

The combination therapy can include one or more LY6H antagonists, e.g.,anti-LY6H antibodies or fragments thereof, formulated with, and/orco-administered with, one or more additional therapeutic agents, e.g.,one or more cytokine and growth factor inhibitors, immunosuppressants,anti-inflammatory agents (e.g., systemic anti-inflammatory agents),anti-fibrotic agents, metabolic inhibitors, enzyme inhibitors, and/orcytotoxic or cytostatic agents, mitotic inhibitors, antitumorantibiotics, immunomodulating agents, vectors for gene therapy,alkylating agents, antiangiogenic agents, antimetabolites,boron-containing agents, chemoprotective agents, hormones, antihormoneagents, corticosteroids, photoactive therapeutic agents,oligonucleotides, radionuclide agents, topoisomerase inhibitors,tyrosine kinase inhibitors, or radiosensitizers, as described in moreherein.

In a particular embodiment, the anti-LY6H antibodies disclosed hereinare used in combination with an anti-cancer agent or an antineoplasticagent. The terms “anti-cancer agent” and “antineoplastic agent” refer todrugs used to treat malignancies, such as cancerous growths. In oneembodiment, the anti-LY6H antibodies or ADCs of the invention areadministered in combination with one or more immune checkpointinhibitors (e.g., antibody or small molecule immune checkpointinhibitors) for the treatment of a cancer. In some embodiments, theimmune checkpoint inhibitor (ICI) is an inhibitor (e.g., an antibody) ofProgrammed Death-Ligand 1 (PD-L1, also known as B7-H1, CD274),Programmed Death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3,2B4, 4-1BB, A2aR, B7H1, B7H3, BTLA, CD2, CD6, CD27, CD28, CD30, CD38,CD39, CD40, CD47, CD70, CD73, CD80, CD86, CD137, CD160, CD166, CD200,CD200R1, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS(inducible T cell costimulator), KIR, LAG3, LAIR1, TREM2, LILRB1,LILRB2, LILRB3, LILRB4, LIGHT, MARCO (macrophage receptor withcollageneous structure), PS (phosphatidylserine), OX-40, SLAM, SIRPA,CSF1R, CD47, SIRPA, TIGHT, TGFβ, VISTA, or any combinations thereof.

In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4,PD-L1, or PD-1 antibody therapy such as, but not limited to Yervoy®(ipilimumab; Bristol-Myers Squibb), Opdivo® (nivolumab; Bristol-MyersSquibb), Keytruda® (pembrolizumab; Merck), and Tecentriq® (atezolizumab;Roche).

In other embodiments, the immune checkpoint inhibitor is an anti-CD38antibody therapy such as isatuximab (Sanofi), Darzalex® (daratumumab;Genmab A/S and Janssen Biotech), MOR202 (MorphoSys AG), and TuskTherapeutics Ltd.'s anti-CD38 monoclonal antibody.

In some embodiments, the checkpoint inhibitor is an antibody or smallmolecule currently undergoing clinical testing, including, for example,an antibody against IDO (Epacadostat and Indoximod and BMS-986205),4-1BB/CD137 (Utomilumab and Urelumab), KIR (Lirilulmab), CD40(CP-870,893), CD27 (Varlilumab), LAG-3 (Relatilimab), MHCII (EftilagimodAlpha).

In one embodiment, the anti-LY6H antibodies or ADCs of the invention areadministered in combination with one checkpoint inhibitor, e.g., ananti-CTLA-4, CD38, PD-L1, or PD-1 antibody. In other embodiments, theanti-LY6H antibodies or ADCs of the invention are administered incombination with more than one checkpoint inhibitor, e.g., an anti-LY6Hantibody or ADC of the invention in combination with an anti-CD38antibody and an anti-PD-L1 antibody, or an anti-LY6H antibody or ADC ofthe invention in combination with an anti-CD38 antibody and an anti-PD-1antibody.

Drug therapy may be used alone, or in combination with other treatmentssuch as surgery or radiation therapy. Several classes of drugs may beused in cancer treatment, depending on the nature of the organ involved.For example, breast cancers are commonly stimulated by estrogens, andmay be treated with drugs which inactive the sex hormones. Similarly,prostate cancer may be treated with drugs that inactivate androgens, themale sex hormone.

Provided herein are methods for treating cancer, e.g., asmall cell lungcancer (SCLC), e.g., classic SCLC or variant SCLC, gastrointestinalstromal tumor (GIST), T cell acute lymphoblastic leukemia (T-ALL),glioblastoma, soft tissue sarcoma, breast cancer, brain and CNS cancer,head and neck cancer, bladder cancer, renal cancer, ovarian cancer,esophageal cancer, gastric cancer, prostate cancer, uterine cancer,endometrial cancer, pancreatic cancer, liver cancer, colorectal cancer,cervical cancer and non small cell lung cancer (NSCLC), or a disorder inwhich LY6H activity is detrimental, in a patient comprisingadministering to the patient an anti-LY6H antibody, or fragment thereof,or an ADC of the invention wherein the combination therapy exhibitssynergy, e.g., therapeutic synergy, in the subject. As used herein,“synergy” or “therapeutic synergy” refers to a phenomenon wheretreatment of patients with a combination of therapeutic agents manifestsa therapeutically superior outcome to the outcome achieved by eachindividual constituent of the combination used at its optimum dose(Corbett, T. H. et al., Cancer Treatment Reports, 66:1187 (1982)). Forexample, a therapeutically superior outcome is one in which the patientseither a) exhibit fewer incidences of adverse events while receiving atherapeutic benefit that is equal to or greater than that whereindividual constituents of the combination are each administered asmonotherapy at the same dose as in the combination, or b) do not exhibitdose-limiting toxicities while receiving a therapeutic benefit that isgreater than that of treatment with each individual constituent of thecombination when each constituent is administered in at the same dosesin the combination(s) as is administered as individual components.

In particular embodiments, the anti-LY6H antibodies or ADCs can beadministered alone or with another anti-cancer agent which acts inconjunction with or synergistically with the antibody to treat thedisease associated with LY6H activity. Such anti-cancer agents include,for example, one or more agents well known in the art (e.g., cytotoxins,chemotherapeutic agents, such as gemcitabine, carboplatin, and 5-Fu,small molecules and radiation) or one or more immune checkpointinhibitor as set forth above. In one embodiment, the one or morechemotherapeutic agent is pemetrexed (Alimta®) and/or platinumchemotherapy, e.g., cisplatin or carboplatin (see e.g., Gandhi et al.New England Journal of Medicine DOI: 10.1056/NEJMoa1801005, Apr. 16,2018).

Other examples of anti-cancer agents include, but are not limited to,Panorex (Glaxo-Welcome), Rituxan (IDEC/Genentech/Hoffman la Roche),Mylotarg (Wyeth), Campath (Millennium), Zevalin (IDEC and Schering AG),Bexxar (Corixa/GSK), Erbitux (Imclone/BMS), Avastin (Genentech) andHerceptin (Genentech/Hoffman la Roche). Examples of anti-cancer agentsthat can be administered in combination with an anti-LY6H antibody orADC of the invention include any one or more of those agents describedabove in Section III (A) of this disclosure.

In one embodiment, the anti-LY6H antibodies or ADCs of the invention areadministered in combination with one or more compound which is capableof decreasing T regulatory cells and/or increasing effector T cell:Tregulatory cell ratio in a subject (see, e.g., Eriksson et al. (2016)Journal of Translational Medicine 14:282). In one embodiment, thecompound is, for example, gemcitabine.

In another embodiment, the anti-LY6H antibodies or ADCs can beadministered in combination with an anti-cancer agent that regulates thetumor micro-environment, including inhibiting the activity or populationof MDSCs and macrophages, such as, for example, CSF-1R antibodies,all-trans retinoic acid, gemcitabine, COX2 inhibitor (SC58236),amino-biphosphonate, phosphodiesterase-5 inhibitor (sildenafil andtadalafil), KIT-specific antibody, nitroaspirin, titerpenoid,25-hydroxyvitamin D3, VEGF-trap, VEGF-specific antibody (e.g., Avastin),doxorubicincyclophosphamide, antagonists for CXCR2 (e.g., S-265610) andCXCR4 (e.g., AMD3100), tyrosine kinase inhibitor (e.g., Sunitinib), andPROK2-specific antibody (see V. Bronte and D. Gabrilovich, Myeloidderived suppressor cells, Nature Rev. Immunology poster, availablethrough www.Biolegend.com).

In another embodiment, the anti-LY6H antibodies or ADCs can beadministered in combination with anti-cancer agents that modulate tumoragiogenesis such as, but not limited to angiostatin, ABX EGF, C1-1033,PKI-166, EGF vaccine, EKB-569, GW2016, ICR-62, EMD 55900, CP358,PD153035, AG1478, IMC-C225 (Erbitux, ZD1839 (Iressa), OSI-774, Erlotinib(tarceva), angiostatin, arrestin, endostatin, BAY 12-9566 andw/fluorouracil or doxorubicin, canstatin, carboxyamidotriozole and withpaclitaxel, EMD121974, S-24, vitaxin, dimethylxanthenone acetic acid,IM862, Interleukin-12, Interleukin-2, NM-3, HuMV833, PTK787, RhuMab,angiozyme (ribozyme), IMC-1C11, Neovastat, marimstat, prinomastat,BMS-275291, COL-3, MM1270, SU101, SU6668, SU11248, SU5416, withpaclitaxel, with gemcitabine and cisplatin, and with irinotecan andcisplatin and with radiation, tecogalan, temozolomide and PEG interferonα1b, tetrathiomolybdate, TNP-470, thalidomide, CC-5013 and withtaxotere, tumstatin, 2-methoxyestradiol, VEGF trap, mTOR inhibitors(deforolimus, everolimus (Afinitor, Novartis PharmaceuticalCorporation), and temsirolimus (Torisel, Pfizer, Inc.)), tyrosine kinaseinhibitors (e.g., erlotinib (Tarceva, Genentech, Inc.), imatinib(Gleevec, Novartis Pharmaceutical Corporation), gefitinib (Iressa,AstraZeneca Pharmaceuticals), dasatinib (Sprycel, Brystol-Myers Squibb),sunitinib (Sutent, Pfizer, Inc.), nilotinib (Tasigna, NovartisPharmaceutical Corporation), lapatinib (Tykerb, GlaxoSmithKlinePharmaceuticals), sorafenib (Nexavar, Bayer and Onyx), phosphoinositide3-kinases (PI3K).

In some embodiments, the antibody or antigen binding portion thereof orthe ADC is administered in combination with IL-6 and/or interferon-gamma(IFN-γ). For example, IL-6 and/or IFN-γ may be administered prior to theantibody or antigen binding portion thereof or the ADC.

In another embodiment, the antibody or antigen binding portion thereofor the ADC is administered in combination with a DNA alkylator (e.g.,cisplatin) and/or a PARP inhibitor.

Other anti-cancer agents include, but are not limited to, thosedisclosed in U.S. Pat. No. 7,598,028 and International Publication No.WO2008/100624, the contents of which are hereby incorporated byreference. One or more anti-cancer agents may be administered eithersimultaneously or before or after administration of an antibody orantigen binding portion thereof.

In particular embodiments of the invention, the anti-LY6H antibodies orADCs described herein can be used in a combination therapy with aninhibitor of NAMPT (see examples of inhibitors in US 2013/0303509;AbbVie, Inc., incorporated by reference herein) to treat a subject inneed thereof. NAMPT (also known as pre-B-cell-colony-enhancing factor(PBEF) and visfatin) is an enzyme that catalyzes the phosphoribosylationof nicotinamide and is the rate-limiting enzyme in one of two pathwaysthat salvage NAD. In one embodiment, anti-LY6H antibodies and ADCsdescribed herein are administered in combination with a NAMPT inhibitorfor the treatment of cancer in a subject.

In particular embodiments, the anti-LY6H antibodies or ADCs describedherein can be used in a combination therapy with SN-38, which is theactive metabolite of the topoisomerase inhibitor irinotecan.

The pharmaceutical compositions may include a “therapeutically effectiveamount” or a “prophylactically effective amount” of an antibody orantibody portion. A “therapeutically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic result. A therapeutically effectiveamount of the antibody or antibody portion may be determined by a personskilled in the art and may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody or antibody portion to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody, or antibody portion, areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic or prophylacticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such an active compound for the treatment of sensitivity inindividuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an ADC, an antibody or antibodyportion is 0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to be notedthat dosage values may vary with the type and severity of the conditionto be alleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

In another aspect, this application provides a method for detecting thepresence of LY6H in a sample in vitro (e.g., a biological sample, suchas serum, plasma, tissue, biopsy). The subject method can be used todiagnose a disorder, e.g., a cancer. The method includes: (i) contactingthe sample or a control sample with the anti-LY6H antibody or fragmentthereof as described herein; and (ii) detecting formation of a complexbetween the anti-LY6H antibody or fragment thereof, and the sample orthe control sample, wherein a statistically significant change in theformation of the complex in the sample relative to the control sample isindicative of the presence of LY6H in the sample.

Given their ability to bind to human LY6H, the anti-human LY6Hantibodies, or portions thereof, (as well as ADCs thereof) can be usedto detect human LY6H (e.g., in a biological sample, such as serum orplasma), using a conventional immunoassay, such as an enzyme linkedimmunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissueimmunohistochemistry. In one aspect, the disclosure provides a methodfor detecting human LY6H in a biological sample comprising contacting abiological sample with an antibody, or antibody portion, and detectingeither the antibody (or antibody portion) bound to human LY6H or unboundantibody (or antibody portion), to thereby detect human LY6H in thebiological sample. The antibody is directly or indirectly labeled with adetectable substance to facilitate detection of the bound or unboundantibody. Suitable detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ³H, ¹⁴C,³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

Alternative to labeling the antibody, human LY6H can be assayed inbiological fluids by a competition immunoassay utilizing rhLY6Hstandards labeled with a detectable substance and an unlabeledanti-human LY6H antibody. In this assay, the biological sample, thelabeled rhLY6H standards and the anti-human LY6H antibody are combinedand the amount of labeled rhLY6H standard bound to the unlabeledantibody is determined. The amount of human LY6H in the biologicalsample is inversely proportional to the amount of labeled rhLY6Hstandard bound to the anti-LY6H antibody. Similarly, human LY6H can alsobe assayed in biological fluids by a competition immunoassay utilizingrhLY6H standards labeled with a detectable substance and an unlabeledanti-human LY6H antibody.

In yet another aspect, this application provides a method for detectingthe presence of LY6H in vivo (e.g., in vivo imaging in a subject). Thesubject method can be used to diagnose a disorder, e.g., aLY6H-associated disorder. The method includes: (i) administering theanti-LY6H antibody or fragment thereof as described herein to a subjector a control subject under conditions that allow binding of the antibodyor fragment to LY6H; and (ii) detecting formation of a complex betweenthe antibody or fragment and LY6H, wherein a statistically significantchange in the formation of the complex in the subject relative to thecontrol subject is indicative of the presence of LY6H.

V. Pharmaceutical Compositions

The disclosure also provides pharmaceutical compositions comprising anantibody, or antigen binding portion thereof, or ADC and apharmaceutically acceptable carrier. The pharmaceutical compositionscomprising antibodies or ADCs are for use in, but not limited to,diagnosing, detecting, or monitoring a disorder, in preventing,treating, managing, or ameliorating of a disorder or one or moresymptoms thereof, and/or in research. In a specific embodiment, acomposition comprises one or more antibodies. In another embodiment, thepharmaceutical composition comprises one or more antibodies or ADCs andone or more prophylactic or therapeutic agents other than antibodies orADCs for treating a disorder in which LY6H activity is detrimental.Preferably, the prophylactic or therapeutic agents known to be usefulfor or having been or currently being used in the prevention, treatment,management, or amelioration of a disorder or one or more symptomsthereof. In accordance with these embodiments, the composition mayfurther comprise of a carrier, diluent or excipient.

The antibodies and antibody-portions or ADCs can be incorporated intopharmaceutical compositions suitable for administration to a subject.Typically, the pharmaceutical composition comprises an antibody orantibody portion and a pharmaceutically acceptable carrier. As usedherein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the antibodyor antibody portion or ADC.

Various delivery systems are known and can be used to administer one ormore antibodies or ADCs or the combination of one or more antibodies anda prophylactic agent or therapeutic agent useful for preventing,managing, treating, or ameliorating a disorder or one or more symptomsthereof, e.g., encapsulation in liposomes, microparticles,microcapsules, recombinant cells capable of expressing the antibody orantibody fragment, receptor-mediated endocytosis (see, e.g., Wu and Wu,J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of administering aprophylactic or therapeutic agent include, but are not limited to,parenteral administration (e.g., intradermal, intramuscular,intraperitoneal, intravenous and subcutaneous), epidural administration,intratumoral administration, and mucosal administration (e.g.,intranasal and oral routes). In addition, pulmonary administration canbe employed, e.g., by use of an inhaler or nebulizer, and formulationwith an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968,5,985,320, 5,985,309, 5,934, 272, 5,874,064, 5,855,913, 5,290, 540, and4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO97/44013, WO 98/31346, and WO 99/66903, each of which is incorporatedherein by reference their entireties. In one embodiment, an antibody,combination therapy, or a composition is administered using AlkermesAIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge,Mass.). In a specific embodiment, prophylactic or therapeutic agents areadministered intramuscularly, intravenously, intratumorally, orally,intranasally, pulmonary, or subcutaneously. The prophylactic ortherapeutic agents may be administered by any convenient route, forexample by infusion or bolus injection, by absorption through epithelialor mucocutaneous linings (e.g., oral mucosa, rectal and intestinalmucosa, etc.) and may be administered together with other biologicallyactive agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents locally to the area in need oftreatment; this may be achieved by, for example, and not by way oflimitation, local infusion, by injection, or by means of an implant,said implant being of a porous or non-porous material, includingmembranes and matrices, such as sialastic membranes, polymers, fibrousmatrices (e.g., Tissuel®), or collagen matrices. In one embodiment, aneffective amount of one or more antibodies antagonists is administeredlocally to the affected area to a subject to prevent, treat, manage,and/or ameliorate a disorder or a symptom thereof. In anotherembodiment, an effective amount of one or more antibodies isadministered locally to the affected area in combination with aneffective amount of one or more therapies (e.g., one or moreprophylactic or therapeutic agents) other than an antibody of a subjectto prevent, treat, manage, and/or ameliorate a disorder or one or moresymptoms thereof.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude, but are not limited to, parenteral, e.g., intravenous,intradermal, subcutaneous, oral, intranasal (e.g., inhalation),transdermal (e.g., topical), transmucosal, and rectal administration. Ina specific embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous, subcutaneous, intramuscular, oral, intranasal, or topicaladministration to human beings. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic such as lignocaine to ease pain at the site of theinjection.

If the method of the disclosure comprises oral administration,compositions can be formulated orally in the form of tablets, capsules,cachets, gel caps, solutions, suspensions, and the like. Tablets orcapsules can be prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinised maizestarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose, or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well-known in the art. Liquid preparations for oraladministration may take the form of, but not limited to, solutions,syrups or suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated for slow release, controlledrelease, or sustained release of a prophylactic or therapeutic agent(s).

The method may comprise administration of a composition formulated forparenteral administration by injection (e.g., by bolus injection orcontinuous infusion). Formulations for injection may be presented inunit dosage form (e.g., in ampoules or in multi-dose containers) with anadded preservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle (e.g., sterile pyrogen-free water)before use.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, compositioncan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

In particular, the disclosure also provides that one or more of theprophylactic or therapeutic agents, or pharmaceutical compositions ispackaged in a hermetically sealed container such as an ampoule orsachette indicating the quantity of the agent. In one embodiment, one ormore of the prophylactic or therapeutic agents, or pharmaceuticalcompositions is supplied as a dry sterilized lyophilized powder or waterfree concentrate in a hermetically sealed container and can bereconstituted (e.g., with water or saline) to the appropriateconcentration for administration to a subject. The antibodies andantibody-portions or ADCs can be administered by a variety of methodsknown in the art, although for many therapeutic applications, thepreferred route/mode of administration is subcutaneous injection,intravenous injection or infusion. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. In certain embodiments, the active compoundmay be prepared with a carrier that will protect the compound againstrapid release, such as a controlled release formulation, includingimplants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein. Having now described the invention in detail, the samewill be more clearly understood by reference to the following examples,which are included for purposes of illustration only and are notintended to be limiting.

EXAMPLES Example 1. LY6H is Differentially and Abundantly Expressed inPrimary Small Cell Lung Cancer (SCLC) Tumors by Proteomics

Experiments were performed to determine LY6H protein expression in SCLCtumors using the following methods.

Methods

TMT10-Plex Analysis of Membrane Proteins from Human Tumors

Primary SCLC tumors, normal adjacent tissue from primary tumors, andnormal lung tissues were acquired as either fresh frozen or in formalinfixed paraffin embedded blocks (FFPE). Samples were enriched usingstandard in-house methods, digested and labeled with TMT. Up to tensamples were combined to create a TMT10-plex, which was fractionated bybRP liquid chromatography. Fractions were subjected to multi-notchLC-MS³ on an Orbitrap Fusion Lumos mass spectrometer.

From 22 SCLC specimens, 867,028 peptides were identified with a falsediscovery rate (FDR) of 0.25%, mapping to 11,465 collapsed proteins withan FDR<2%. This filtered dataset was further processed for high qualityquantification events in an automated fashion using standard in-housesoftware described, e.g., in McAlister, G. C. et al. MultiNotch MS3enables accurate, sensitive, and multiplexed detection of differentialexpression across cancer cell line proteomes (McAlister, G. C., et al.Anal Chem 86, 7150-7158, 2014). iBAQ (intensity-based absolutequantification) data were presented.

Results

To identify cell surface targets that are differentially expressed insmall cell lung cancer (SCLC), 22 primary small cell lung cancer (SCLC)specimens, 16 chemotherapy refractory specimens, 5 normal adjacentspecimens, and 10 normal lung specimens were obtained and profiled byproteomics. LY6H was observed to be highly expressed in 68% of SCLCpatients (FIG. 1). The average copy number expression of LY6H in humanSCLC tumors is approximately 230,000 copies per cell, suggesting thatLY6H is abundantly expressed in SCLC tumors. DLL3, which is currentlybeing pursued in clinical trials as an antibody-drug-conjugate (ADC) inSCLC, was used as a reference (Table 1)

TABLE 1 LY6H is abundantly expressed in SCLC tumors Cell Chemo- PTMs %pat > Avg % PDXs Line refractory in Target 5(FC) Copy# (RNA) Modelsexpression Cancer LY6H 68% 230,000 17% Limited ✓ n/a DLL3 91% 4,500 76%Yes ✓ n/a % pat > 5: Percentage of patients with a >5 fold-changecompared to vital organs. Avg Copy#: Average number of Molecules/tumorlysate determined using targeted proteomics (estimating 15,000 cells = 1ug of input protein). % PDXs: Percentage of CrownBio SCLC PDX models > 4on a log2 FPKM scale for given target. Chemo-refractory expression: ✓ =expression of protein shows similar profile to primary tumors. PTMs InCancer: Post-translational modifications reported in PhosPhoSitePlus(phosphosite.org) in cancer cell lines or tumors.

Example 2. Generation of Human Monoclonal Antibodies Against the LY6HExtracellular Domain

Experiments were performed to generate fully human antibodies againstLY6H-ECD using the following methods.

Methods

Immunizations in Humanized Mice

Monoclonal antibodies were obtained by immunizing Harbour H2L2 mice witheither recombinant human LY6H-ECD or BaF3 cell lines expressingfull-length LY6H tagged with MYC-DDK at the C-terminus. H2L2 mice wereengineered with the capacity to produce human immunoglobulins at thevariable region. The mice received 5 rounds of either recombinantprotein or cells by intraperitoneal injection (IP) and allowed to restfor one month. Then, the mice were boosted 4 and 2 days prior to fusionof the spleen with rabbit splenocytes expressing full length LY6H orrecombinant protein of the extracelluar domain (ECD) of LY6H. HumanLY6H-ECD recombinant protein was expressed in EBNA293 cells.

Recombinant LY6H Cloning

Human LY6H cDNA was purchased from Origene (RC205136, Rockville, Md.)and named BBP. The encoded protein aligns 100% with GenBank LY6H_HUMAN.Macaca fascicularis, rat, and mouse LY6H cDNA, were synthesized fromGen9 (Cambridge, Mass.).

Ectodomains of human, Macaca fascicularis, rat and mouse were cloned byeither PCR (human) or synthetic genes (Macaca fascicularis, rat, mouse).The synthetic genes were based on GenBank sequences (Table 2). All DNAsequences were cloned into appropriate CMV-based expression vectors withnon-native signal peptides and C-terminal histidine tags forpurification.

TABLE 2 Source of LY6H protein sequences GenBank Protein SpeciesReference Sequence SEQ ID NO Homo sapiens LY6H_HUMANMLPAAMKGLGLALLAVLLCSAPAHGL 123 (human) NP_002338.3WCQDCTLTTNSSHCTPKQCQPSDTVCA SVRITDPSSSRKDHSVNKMCASSDFVKRHFFSDYLMGFINSGILKVDVDCCEKDLC NGAAGAGHSPWALAGGLLLSLGPALL WAGP MacacaLY6H_MACFA MLPAAMKGLGLALLAVLLCSAPAHGL 124 fascicularis Sp_Q4R5M8WCQDCTLTTNSSHCTPKQCQPSDTVCA SVRITDPSSSRKDHSVNKMCASSCDFVKRHFFSDYLMGFINSGILKVDVDCYEKDL CNGVAGAGHSPWALAGGLLLSLGPALL WAGPRattus novegicus NP_001128311.1 MLPAAMKSLGLALLALLLCPSPAHGLW 125 (rat)CQDCTLANSSHCAPKQCQPTDTVCASV RITDPSSSRKDHSVNKMCASSCDFVKRHFFSDYLMGFINSGILKDVDCCEKDLCN GASAAGRSPWALAGGLLLSLGPALLW AGP Mus musculusLY6H_MOUSE MLPAAMKSLGLALLALLLCPSPAHGLW 126 (mouse) NP_035967.1CQDCTLANSSHCAPKQCQPTDTVCASV RITDPSSSRKDHSVNKMCASSCDFVKRHFFSDYLMGFINSGILKVDVDCCEKDLC NGASVAGRSPWALAGGLLLSLGPALL WAGP

The retroviral MSCV construct was used to express full-length human LY6Hprotein on the surface of HEK-293T, BaF3 cells, and rabbit splenocytes(see Table 3). Mouse, rat and Macaca fascicularis LY6H were alsoexpressed on the surface of HEK-293T.

TABLE 3 LY6H cell-surface expression vectors Plasmid name SpeciesSequence feature Comment BBP491 Human M1-P140 (Plus Myc-DDK) Fullconstruct BBP 492/ macaca M1-P140 (Plus Myc-DDK) Full construct 598Fascicularis BBP493 Rat M1-P139 (Plus Myc-DDK) Full construct BBP494Mouse M1-P160 (Plus Myc-DDK) Full construct

Cloning VH and VL Sequences from Hybridomas

For determination of CDR sequences, total RNA was isolated fromhybridoma cells using an RNeasy® kit (Qiagen, Hilden, Germany). Firstand second-strand cDNA synthesis was performed using a OneTaq® One-StepRT-PCR kit (New England BioLabs, Ipswich, Mass.). Several primer setswere used (Table 4). PCR products were separated by agaroseelectrophoresis and fragments were excised and purified by a QIAquick®gel extraction kit (Qiagen, Hilden, Germany). Fragments were cloneddirectly into expression vectors with BspQI (New England BioLabs,Ipswich, Mass.) by Golden Gate cloning techniques. Four colonies fromeach reaction were scaled up for miniprep-scale plasmid purification bySequeMid® DNA Purification Kit (Aline Biosciences, Woburn, Mass.).

TABLE 4 Oligonucleotide Sequences SEQ ID Oligonucleotide NO:Oligonucleotide Sequence Purpose  97ATAGCTCTTCAGGGaccATGAARCAYCTGTGGTTCTTCCT IGHV4 leader  98ATAGCTCTTCAGGGaccATGGACATACTTTGTTCCACGC IGHV2 leader  99ATAGCTCTTCAGGGaccATGGACACACTTTGCTACACAC IGHV2-26 leader 100ATAGCTCTTCAGGGaccATGTCTGTCTCCTTCCTCATCT IGHV6 leader 101ATAGCTCTTCAGGGaccATGGACTGGACCTGGAGVATC IGHV1 leader 102ATAGCTCTTCAGGGaccATGGACTGGATTTGGAGGRTC IGHV1-58 leader 103ATAGCTCTTCAGGGaccATGGACTGCACCTGGAGGATC IGHV1-24 leader 104ATAGCTCTTCAGGGaccATGGACTGGACCTGGAGGKTC IGHV1-69/1-46/ 7-4-1 leader 105ATAGCTCTTCAGGGaccATGGAGTTKGGRCTGAGCTGG IGHV3 leader 106ATAGCTCTTCAGGGaccATGGAGTTTKGGCTKAGCTGG IGHV3-53/3-49 leader 107ATAGCTCTTCAGGGaccATGGAACTGGGGCTCCGCTGG IGHV3-21 leader 108ATAGCTCTTCAGGGaccATGGARTTGGGGCTGWGCTGG IGHV3-48/3-7 leader 109ATAGCTCTTCAGGGaccATGGGGTCAACCGCCATCCTC IGHV5 leader 110ATAGCTCTTCAGGGaccATGGACATGAGGGTSCCYGCTCAGCTC IgkV1a leader 111ATAGCTCTTCAGGGaccATGGACATGAGRGTCCTCGCTCAGCTC IgkV1b leader 112ATAGCTCTTCAGGGaccATGGAAGCCCCAGCDCAGCTTCTC IgkV3 leader 113ATAGCTCTTCAGGGaccATGGAAACCCCAGCGCAGCTTCTC IgkV3-20 leader 114ATAGCTCTTCAGGGaccATGGTGTTGCAGACCCAGGTCTTC IgkV4 leader 115ATAGCTCTTCAGGGaccATGGGGTCCCAGGTTCACCTCCTC IgkV5 leader 116ATAGCTCTTCAGGGaccATGAGGCTCCYTGCTCAGCTCCTG IgkV2 leader 117ATAGCTCTTCTTCGTTTGATCTCCASCTTGGTC Kappa FW4 118ATAGCTCTTCTTCGTTTAATCTCCAGTCGTGTC Kappa FW4 119ATAGCTCTTCTGGCTGAGGAGACGGTGACC Heavy FW4 120ATAGCTCTTCATGTGACGCTGTTGTGACTCAGGA VL-FOR L1 121ATAGCTCTTCATGTGACCYTGTGCTCACTCAGTC VL-FOR L2 122GATGCTCTTCTGGGCTGGCCTAGGACAGTCAMCYTGG VL-REV L

Identification of Functional, Recombinant VH and VL Sequences

For each hybridoma, each plasmid was sent for Sanger Sequencing. Theseplasmids were subjected to DNA sequence determination and analysis.Unique recombinant heavy chains were paired with unique recombinantlight chains. These plasmid pairs were transfected into CHO cells in24-well plates. Ten days later conditioned medium from each pairing wasscreened by FLOW™ or Octet™ for binding to LY6H.

Transient Expression System of Medium Scale Antibody Production orRecombinant Proteins

The LY6H recombinant proteins and anti-LY6H antibodies were expressed inChinese hamster ovary (CHO) cells in a 100 ml volume flask usingrecommended transfection and media components of the ExpiCHO™ system(Invitrogen, Carlsbad, Calif.). Cell culture supernatants were harvested14 days post-transfection, centrifuged, and filtered (0.22 um).

Purification of Recombinant His-Tagged Proteins

Conditioned medium from CHO cell cultures was clarified, filtered, andloaded onto an ÄKTAprime Plus™ system with a 5 mL HisTrap™ FF column (GEHealthcare). Fractions were collected, analyzed by SDS-PAGE, pooled, anddialyzed against PBS.

Antibody Purification

Conditioned medium from CHO cell cultures was clarified, filtered, andpurified by loading onto an ÄKTA Pure™ system with a 5 mL MabSelectSuRe® column (GE Healthcare). Antibodies were eluted with 100 mMglycine, pH 3.5 and neutralized with 1M Tris-Cl, pH 8.5.

Recombinant Antibody Analyses

Concentration:

Concentration of recombinant antibodies was determined on a FortebioOctet Red™ (Pall ForteBio, Fremont, Calif.) instrument using Protein Atips and a human IgG1 antibody for the standard curve.

Purity Testing by SDS-PAGE:

Purity testing was performed by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) of reduced and non-reduced samples.Samples (10 ug) were mixed with loading buffer (+/−β-mercaptoethanol),heated, and electrophoresed on a 4-20% gel (Invitrogen, Carlsbad,Calif.). Bands were visualized by Coomassie InstantBlue™ (Expedeon, SanDiego, Calif.) staining.

Purity Testing by Endotoxin:

Endotoxin concentrations were measured by the Limulus amoebocyte lysate(LAL) kinetic turbidometric method using the Endosafe-PTS system(Charles River Laboratories, Wilmington, Mass.).

Purity Testing by HPLC-SEC:

Samples were screened for aggregation or other forms of antibody on a1260 Infinity System™ (Agilent, Santa Clara, Calif.) with a TSKgelUltraSW Aggregate Guard™ column and HPLC column (Tosoh Bioscience).Samples and standards were detected by absorbance at 280 nm. Comparisonagainst the standard curve provided the molar mass of sample components.

Affinity:

The affinity of antibodies to various recombinant LY6H protein wasdetermined on an Octet Red™ instrument. After loading reagents into a96-well plate, the Octet Red with Protein A-conjugated biosensors wasprogrammed as follows: 30 seconds for baseline #1; 120 seconds toimmobilize the antibody; 30 seconds for baseline #2; 300 seconds forassociation of antibody to recombinant LY6H; and 300-600 seconds fordissociation of recombinant LY6H from the antibody.

Binding Competition Binning:

Binding competition among different antibodies was determined using areal-time, interferometry assay on an Octet Red™ instrument with ProteinA-conjugated biosensors. To assess whether two antibodies competed forbinding to a recombinant LY6H protein, the assay was performed asfollows. Protein A biosensors were first submerged into wells containing10 ug/mL of individual monoclonal antibodies for 5 minutes. Followingthe capture step, the biosensors were dipped briefly (15 seconds) intobuffer and then any unoccupied sites on the biosensor were saturated bysubmerging them for 5 minutes into wells containing 100 ug/mL of anirrelevant monoclonal antibody. The Octet biosensors were then dippedbriefly (15 seconds) in buffer before immersion for 1 minute into wellscontaining recombinant LY6H. The biosensors were dipped briefly (15seconds) in buffer before immersion for 1 minute into wells containing asecond recombinant antibody.

For the control case where the second antibody was the same as thefirst, there was no increase in signal, because there was no additionalbinding to the recombinant target.

For the control case where buffer was used instead of the firstantibody, no recombinant target bound the non-quenching antibody on thebiosensor and no second antibody bound the biosensor.

For cases where a boost in signal was seen with the second antibody, thetwo antibodies were determined not to compete.

For cases where no boost in signal was seen with the second antibody,the two antibodies were determined to compete for binding.

Immunofluorescence (IF) Based High Content Screening (HCS)

High content immunofluorescence was used to identify wells that containimmunoglobulin that preferentially bound cells expressing LY6H. Briefly,293 cells, 293-hLY6H, and H446 cells seeded 24 hours before the assaywere incubated for 60 minutes at 37° C. with hybridoma supernatantdiluted 2-fold in DMEM+10% fetal bovine serum (FBS). After incubation,cells were fixed in 4% formaldehyde, washed with PBS, permeabilized with0.3% Triton-X-100, and labeled with anti-rat Alexa 488 (at hybridomastage) or anti-human Alexa 488 secondary antibodies (with recombinantLY6H antibodies) for 1 hour at room temperature. Unbound secondaryantibody was removed with PBS washes, and cells were stained with DNAdye (propidium iodide and Hoechst 33342).

Potential hits were initially identified via low-resolution, highthroughput screening using a TTP Labtech Acumen eX3 (TTP Labtech,Cambridge, Mass.), quantifying the fluorescence differential for eachsample on both positive and negative cell lines. Those hits weresubsequently verified and the subcellular localization of each samplewas characterized using a Thermo ArrayScan VTi (Thermo FisherScientific, Waltham, Mass.) to obtain high-resolution images of bothcell lines.

Flow Cytometry

Staining for flow cytometry was performed in 1× cold PBS with 0.5% BSA.Primary antibodies (1 ug/ml) were incubated with live cells on ice for30 minutes, after a brief wash, cells were incubated with Alexa Fluro®488-conjugated anti-human IgG secondary antibody @1:1000 (709-546-149,Jackson ImmunoResearch). Acquisition of the data was performed on aMACSQuant® Flow Cytometers (Miltenyi Biotec) and analyzed with FlowJosoftware.

Results

Fully human antibodies against LY6H were generated by hybridomaprocedures. Briefly, H2L2 mice were immunized with either recombinanthuman Ly6H-ECD or BaF3 cells overexpressing LY6H and boosted with rabbitsplenocytes expressing full length human LY6H or with ECD of LY6Hrecombinant protein. Splenocytes were fused with the mouse myeloma cellline X63-Ag8.653. Clones from H2L2 mice were identified byimmunofluorescence (IF) based high content screening (HCS) on 293 cellsoverexpressing hLY6H, ratLy6H, and parental 293 cells not expressingLY6H, as well as endogenous small cell lung cancer (SCLC) cell line,H446. From H2L2 mice receiving either whole cell or recombinanthLY6H-ECD immunizations, over 1000 hits were identified that showed MFIratio >10 in 293-human LY6H vs. parental 293 cells, and bind endogenousH446 SCLC cell line. 270 hits were cryopreserved and stored in liquidnitrogen. Approximately 100 clones were selected for molecular cloning.Based on unique CDR3 sequences from the heavy chain variable domain, 10antibodies from 8 families were chosen and confirmed by flow cytometry(MACSQuant® www.miltenybiotec.com) on the following cell lines. Thesmall-cell-lung cancer cells (SCLC) included H446, DMS79, and H526cells. The T-cell acute lymphoblastic leukemia (T-ALL) cancer cellsincluded MOLT3 and KE-37 cells. Binding properties of a representativeantibody clone 12G7 is shown in FIG. 2, and summarized in Table 5.

TABLE 5 Summary of Anti-Ly6H antibodies binding to endogenous cell linesby flow cytometry Ab families Clone ID H446 H69 DMS79 MOLT3 KE-37 EOL1 123F3_19B2 (++++) (+/−) (+/++) (+/++) (−) 2 24A10_19B3 (++++) (−) (++)(+/++) (−) 34H6_20A1 (++++) (+/−) (+++) (+++) (+++) (−) 3 26F3_19B5(+++) (+/−) (++) (+/++) (−) 4 11D9_19D5 (++++) (+) (+++) (+++) (+++) (+)5 12G7_19A2 (++++) (−) (+++) (+++) (++++) (−) 6 1F8_25B6 (++++) (−)(+++) (+++) (+++) (−) 2B11_25D1 (++++) (−) (+++) (++/+++) (+++) (−) 710B10_25A1 (++++) (+/−) (+++) (+++) (++++) (+/−) 8 6D11_25D6 (++++)(+/−) (+++) (+++) (++++) (+/−) (−) No binding (+) Weak binding (++)Moderate binding (+++) Strong binding (++++) Very strong bindingComplete amino acid sequences of the heavy and light chains from theseantibodies are set forth in Table 6.

TABLE 6 CDR and variable region sequencs of human antibodies SEQ IDClone Protein NO: Name Domain Sequences  1 6D11 VHQVQLQQWGAGLFKPSETLSLTCAVYGGSFSGSLWSWIRQPPGKGLEWIGEINHSGSTNYTPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARGRHIVVVTAIHSPFDYWGQGTLVTVSS  2 6D11 CDR-H1 GGSFSGSLWS  3 6D11 CDR-H2EINHSGSTNYTPSLKS  4 6D11 CDR-H3 GRHIVVVTAIHSPFDY  5 6D11 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIFAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSA PRTFGQGTKLEIK  6 6D11CDR-L1 RASQGISNYLA  7 6D11 CDR-L2 AASTLQS  8 6D11 CDR-L3 QKYNSAPRT  911D9 VH QVQLQESGPGLVKPSGTLSLTCTVSGGSISSSSWWSWVRLPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCGGVRGVVMAFDIWGQGTMVTVSS 10 11D9 CDR-H1 GGSISSSSWWS 11 11D9 CDR-H2EIYHSGSTNYNPSLKS 12 11D9 CDR-H3 VRGVVMAFDI 13 11D9 VLDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYST PLTFGGGTKVEIK 14 11D9CDR-L1 RASQSISSYLN 15 11D9 CDR-L2 AASSLQS 16 11D9 CDR-L3 QQSYSTPLT 1723F3 VH QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARVDILTGGNFDYWGQGTLVTVSS 18 23F3 CDR-H1 GGSISSSNWWS 11 23F3 CDR-H2EIYHSGSTNYNPSLKS 19 23F3 CDR-H3 VDILTGGNFDY 20 23F3 VLDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYST PITFGQGTRLEIK 14 23F3CDR-L1 RASQSISSYLN 15 23F3 CDR-L2 AASSLQS 21 23F3 CDR-L3 QQSYSTPIT 2212G7 VH QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYY CAKRWELGAFDIWGQGTMVTVSS23 12G7 CDR-H1 GGSFSGYYWS 24 12G7 CDR-H2 EINHSGSTYYNPSLKS 25 12G7 CDR-H3RWELGAFDI 26 12G7 VL DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSA PRTFGQGTKLEIK  6 12G7CDR-L1 RASQGISNYLA  7 12G7 CDR-L2 AASTLQS  8 12G7 CDR-L3 QKYNSAPRT 2722A5 VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGRGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLWFGESRGGMDVWGQGTTVTVSS 28 22A5 CDR-H1 GFTFSSYGMH 29 22A5 CDR-H2VIWYDGSNKYYADSVKG 30 22A5 CDR-H3 DLWFGESRGGMDV 31 22A5 VLDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM QALQTPHTFGQGTKVEIK 3222A5 CDR-L1 RSSQSLLHSNGYNYLD 33 22A5 CDR-L2 LGSNRAS 34 22A5 CDR-L3MQALQTPHT 35 26F3 VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGRGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRFGELLPFDYWGQGTLVTVSS 28 26F3 CDR-H1 GFTFSSYGMH 29 26F3 CDR-H2VIWYDGSNKYYADSVKG 36 26F3 CDR-H3 RFGELLPFDY 37 26F3 VLDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM QALQTPWTFGQGTKVEIK 3226F3 CDR-L1 RSSQSLLHSNGYNYLD 33 26F3 CDR-L2 LGSNRAS 38 26F3 CDR-L3MQALQTPWT 39 35H10 VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGQWLVQGYFDYWGQGTLVTVSS 28 35H10 CDR-H1 GFTFSSYGMH 29 35H10 CDR-H2VIWYDGSNKYYADSVKG 40 35H10 CDR-H3 DGGQWLVQGYFDY 41 35H10 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSY SRTFGQGTKVEIK 42 35H10CDR-L1 RASQSISSWLA 43 35H10 CDR-L2 KASSLES 44 35H10 CDR-L3 QQYNSYSRT 451F8 VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWYDGSYKYYADSVKGRFTISRDNSKNTLYLQMNSLRTEDTAVYYCARSIVVVTATLDYWGQGTLVTVSS 46 1F8 CDR-H1 GFTFSNYGMH 47 1F8 CDR-H2VIWYDGSYKYYADSVKG 48 1F8 CDR-H3 SIVVVTATLDY 49 1F8 VLEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQHYNNW PPLTFGGGTKLEIK 50 1F8CDR-L1 RASQSVSSNLA 51 1F8 CDR-L2 GASTRAT 52 1F8 CDR-L3 QHYNNWPPLT 532B11 VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRTEDTAVYYCARSIVVVTATLDYWGQGTLVTVSS 46 2B11 CDR-H1 GFTFSNYGMH 29 2B11 CDR-H2VIWYDGSNKYYADSVKG 48 2B11 CDR-H3 SIVVVTATLDY 49 2B11 VLEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQHYNNW PPLTFGGGTKLEIK 50 2B11CDR-L1 RASQSVSSNLA 51 2B11 CDR-L2 GASTRAT 52 2B11 CDR-L3 QHYNNWPPLT 5424A10 VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSIVVVVAVLDYWGQGTLVTVSS 28 24A10 CDR-H1 GFTFSSYGMH 29 24A10 CDR-H2VIWYDGSNKYYADSVKG 55 24A10 CDR-H3 SIVVVVAVLDY 56 24A10 VLEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNW PPITFGQGTRLEIK 50 24A10CDR-L1 RASQSVSSNLA 51 24A10 CDR-L2 GASTRAT 57 24A10 CDR-L3 QQYNNWPPIT 5834H6 VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSIVVVTGFGDYWGQGTLVTVSS 28 34H6 CDR-H1 GFTFSSYGMH 29 34H6 CDR-H2VIWYDGSNKYYADSVKG 59 34H6 CDR-H3 SIVVVTGFGDY 56 34H6 VLEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNW PPITFGQGTRLEIK 50 34H6CDR-L1 RASQSVSSNLA 51 34H6 CDR-L2 GASTRAT 57 34H6 CDR-L3 QQYNNWPPIT 6010B10 VH QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWHWIRQPPGKGLEWIGEINHSESTKYNPSLKSRVTISVDTSKNQFSLKLSSVTGADTAVYYCARGQHIVVVTDSLGDYWGQGTLVTVSS 61 10B10 CDR-H1 GGSFSGYYWH 62 10B10 CDR-H2EINHSESTKYNPSLKS 63 10B10 CDR-H3 GQHIVVVTDSLGDY 26 10B10 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSA PRTFGQGTKLEIK  6 10B10CDR-L1 RASQGISNYLA  7 10B10 CDR-L2 AASTLQS  8 10B10 CDR-L3 QKYNSAPRT 6412G7- VH QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEW S54AIGEINHAGSTYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYY CAKRWELGAFDIWGQGTMVTVSS23 12G7- CDR-H1 GGSFSGYYWS S54A 65 12G7- CDR-H2 EINHAGSTYYNPSLKS S54A 2512G7- CDR-H3 RWELGAFDI S54A 26 12G7- VLDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLL S54AIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSA PRTFGQGTKLEIK  6 12G7-CDR-L1 RASQGISNYLA S54A  7 12G7- CDR-L2 AASTLQS S54A  8 12G7- CDR-L3QKYNSAPRT S54A 66 12G7- VHQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEW N52QIGEIQHSGSTYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYY CAKRWELGAFDIWGQGTMVTVSS23 12G7- CDR-H1 GGSFSGYYWS N52Q 67 12G7- CDR-H2 EIQHSGSTYYNPSLKS N52Q 2512G7- CDR-H3 RWELGAFDI N52Q 26 12G7- VLDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKL N52QLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYN SAPRTFGQGTKLEIK  6 12G7-CDR-L1 RASQGISNYLA N52Q  7 12G7- CDR-L2 AASTLQS N52Q  8 12G7- CDR-L3QKYNSAPRT N52Q 68 6D11- VHQVQLQQWGAGLFRPSETLSLTCAVYGGSFSGSLWSWIRQPPGRGLE 41B4WIGEINHAGSTQYTPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARGRHIVVVTAIHSPFDYWGQGTLVTVSS  2 6D11- CDR-H1 GGSFSGSLWS 41B4 696D11- CDR-H2 EINHAGSTQYTPSLKS 41B4  4 6D11- CDR-H3 GRHIVVVTAIHSPFDY 41B4 5 6D11- VL DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKL 41B4LIFAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYN SAPRTFGQGTKLEIK  6 6D11-CDR-L1 RASQGISNYLA 41B4  7 6D11- CDR-L2 AASTLQS 41B4  8 6D11- CDR-L3QKYNSAPRT 41B4 70 10B10- VHQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWHWIRQPPGKGLE S54AWIGEINHAESTKYNPSLKSRVTISVDTSKNQFSLKLSSVTGADTAVYYCARGQHIVVVTDSLGDYWGQGTLVTVSS 61 10B10- CDR-H1 GGSFSGYYWH S54A 7110B10- CDR-H2 EINHAESTKYNPSLKS S54A 63 10B10- CDR-H3 GQHIVVVTDSLGDY S54A26 10B10- VL DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKL S54ALIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYN SAPRTFGQGTKLEIK  6 10B10-CDR-L1 RASQGISNYLA S54A  7 10B10- CDR-L2 AASTLQS S54A  8 10B10- CDR-L3QKYNSAPRT S54AThe nucleotide (DNA) sequences of the heavy and light chains from theantibodies described in Table 6 are set forth in Table 7.

TABLE 7 Variable region DNA sequencs of human antibodies SEQ ID CloneProtein NO: Name Domain DNA Sequences 72 6D11 VHCAGGTGCAGCTACAGCAATGGGGCGCAGGACTGTTTAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTCCCTCTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACACCCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCTCTGAAGCTGACCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGGCCGGCATATTGTGGTGGTGACTGCTATCCATTCGCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 73 6D11 VLGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTTTGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCCTCGGACGTTCGGCCAAGGGACCAAGCTGGAGATCAAA 74 11D9 VHCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGCTGGTGGAGTTGGGTCCGCCTGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTATCATAGTGGGAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTGGACAAGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTCTATTACTGTGGAGGGGTTCGGGGAGTTGTGATGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCA 75 11D9 VLGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGTTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 76 23F3 VHCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCCTCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAGTAGTAACTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTATCATAGTGGGAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACAAGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGTCGATATTTTGACTGGTGGTAACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 77 23F3 VLGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA 78 12G7 VHCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTTTATTACTGTGCGAAAAGATGGGAGCTTGGTGCTTTTGATATCTGGGGCCAAGG GACAATGGTCACCGTCTCCTCA79 12G7 VL GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCCTCGGACGTTCGGCCAAGGGACCAAGCTGGAGATCAAA 80 22A5 VHCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTATGGTTCGGGGAGTCCCGGGGCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 81 22A5 VLGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCACACTTTTGGCCAGGGGACCAAGGTGGA GATCAAA 82 26F3 VHCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGGAGGTTCGGGGAGTTATTGCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 83 26F3 VLGACATCCAGTTGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAA 84 35H10 VHCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGAGGGCAGTGGCTGGTACAAGGCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 85 35H10 VLGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCTCGGACGTTCGGCCAAGGGACCAAGGTGGAGATCAAA 86 1F8 VHCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTTATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAACCGAGGACACGGCTGTATATTATTGTGCGAGGTCTATTGTGGTGGTGACTGCTACTCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 87 1F8 VLGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCACTATAATAACTGGCCTCCCCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAA 88 2B11 VHCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAACCGAGGACACGGCTGTGTATTACTGTGCGAGGTCTATTGTGGTGGTGACTGCTACTCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 87 2B11 VLGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCACTATAATAACTGGCCTCCCCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAA 89 24A10 VHCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAAGTATTGTAGTGGTGGTAGCTGTCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 90 24A10 VLGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA 91 34H6 VHCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGGTCTATTGTGGTGGTGACTGGATTCGGGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 90 34H6 VLGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA 92 10B10 VHCAGGTGCAGTTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGCACTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGAAAGCACCAAGTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGGCGCGGACACGGCTGTGTATTACTGTGCGAGAGGCCAACATATTGTGGTGGTGACTGATTCTCTGGGGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 79 10B10 VLGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCCTCGGACGTTCGGCCAAGGGACCAAGCTGGAGATCAAA 93 12G7- VHCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGA S54AGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATgcTGGAAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTTTATTACTGTGCGAAAAGATGGGAGCTTGGTGCTTTTGATATCTGGGGCCAAGG GACAATGGTCACCGTCTCCTCA79 12G7- VL GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG S54AAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCCTCGGACGTTCGGCCAAGGGACCAAGCTGGAGATCAAA 94 12G7- VHCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGA N52QGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCcAgCATAGTGGAAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTTTATTACTGTGCGAAAAGATGGGAGCTTGGTGCTTTTGATATCTGGGGCCAAGG GACAATGGTCACCGTCTCCTCA79 12G7- VL GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG N52QGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCCTCGGACGTTCGGCCAAGGGACCAAGCTGGAGATCAAA 95 6D11- VHCAGGTGCAGCTACAGCAATGGGGCGCAGGACTGTTTAAGCCTTCGG 41B4AGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTCCCTCTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATGCTGGAAGCACCCAGTACACCCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCTCTGAAGCTGACCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGGCCGGCATATTGTGGTGGTGACTGCTATCCATTCGCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC CTCA 73 6D11- VLGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG 41B4GAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTTTGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCCTCGGACGTTCGGCCAAGGGACCAAGCTGGAGATCAAA 96 10B10- VHCAGGTGCAGTTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGG S54AAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGCACTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATGCTGAAAGCACCAAGTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGGCGCGGACACGGCTGTGTATTACTGTGCGAGAGGCCAACATATTGTGGTGGTGACTGATTCTCTGGGGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 79 10B10- VLGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG S54AGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCCTCGGACGTTCGGCCAAGGGACCAAGCTGGAGATCAAA

Some antibody sequences with a nonconsensus N-linked glycosylation sitehave been modified at either the Asn site or the Ser/Thr site. Wherepossible, the Asn or Ser/Thr codons can be mutated back to the germlinesequence. In addition, replacing the Asn with Gln or similar amino acidand the Ser or Thr with a similar or smaller amino acid offer areasonable chance of success.

In one case, antibody 12G7 with a dissociation constant (KD) value of 4nM, contained a non-consensus N-linked glycosylation site in CDR2 of theVH sequence. This heavy chain was engineered to contain a Ser54Alamutation. The new heavy chain plasmid was paired with the original lightchain sequence and transfected into CHO cells. The antibody was screenedfor expression and affinity for human LY6H. The 12G7-S54A antibody had aKD value of 4 nM.

In a second case, antibody 10B10 with a dissociation constant (KD) valueof 47 nM, contained a non-consensus N-linked glycosylation site in CDR2of the VH sequence. This heavy chain was engineered to contain aSer54Ala mutation. The new heavy chain plasmid was paired with theoriginal light chain sequence and transfected into CHO cells. Theantibody was screened for expression and affinity for human LY6H. The10B10-S54A antibody had a KD value of 42 nM.

In a third case, antibody 6D11 with a dissociation constant (KD) valueof 22 nM, contained two non-consensus N-linked glycosylation sites inCDR2 of the VH sequence. This heavy chain was engineered to containSer54Ala or Ser54A1a/Asn58Gln or Ser54A1a/Thr60Ala mutations. The newheavy chain plasmids were paired with the original light chain sequenceand transfected into CHO cells. The antibody was screened for expressionand affinity for human LY6H. The 6D11-41B4 antibody containing theSer54A1a/Asn58Gn mutations had a KD value of 25 nM.

Affinity constant values (KD) of several anti-LY6H antibodies are setforth in Table 8.

TABLE 8 Affinity constant values of antibodies to human LY6H Antibody KD(nM) 12G7 4 12G7-S54A 4 6D11 22 6D11-41B4 25 11D9 31 1F8 33 10B10 4710B10-S54A 42

Example 3. Antigen Density of LY6H in SCLC Cell Lines

Experiments were performed to determine LY6H antigen density in SCLCcell lines. The following methods were used.

Methods

Tissue Culture and Cell Lines

Human SCLC cancer cell lines H446, DMS79, NCI-H526, H69, NCI-H220,NCI-H1092, NCI-H2141, CORL47, and CORL95 were obtained from AmericanType Culture Collection (ATCC). Human leukemia cell lines MOLT3 andKE-37 were purchased from DSMZ. NCI-H1092 and NCI-H2141 cells weremaintained in HITS medium. Other cell lines were maintained in RPMI-1640medium (Invitrogen) with 10% fetal bovine serum (FBS) (Sigma).

Antigen Density Measurement

The antigen density of LY6H on the cell surface of cancer cell lines wasquantified using BD Quantibrite™ Beads PE fluorescence Quantitation kit(BD Bioscience, Cat. #340495) and a human LY6H antibody (12G7)conjugated to the fluorochrome PE.

Results

PE conjugated anti-LY6H antibody (12G7) was chosen to evaluate antigendensity in SCLC, and T-ALL (MOLT3, KE-37) cell lines, with H69 cell lineas a negative control. Cell surface LY6H density is estimated at0-250,000 sites per cell for the panel of cancer cell lines tested, withthe highest surface expression level in H446 cells (Table 9).

TABLE 9 Antigen density of LY6H in a panel of cancer cell lines. Celllines Cell Surface Ag density H446 251543 DMS79 39203 H526 10083 CORL4710986 CORL95 19101 CORL279 768 NCI-H220 36983 NCI-H1092 44286 NCI-H214116193 H69 0 MOLT3 12539 KE-37 24811

Example 4. Binding of Anti-LY6H Monoclonal Antibodies to LY6H Orthologs

Experiments were performed to determine the binding of anti-LY6H humanmonoclonal antibodies to LY6H in different species using the followingmethods.

Methods

Tissue Culture and Cell Lines

293T cell line was purchased from American Type Culture Collection(ATCC). 293T cells expressing human, Macaca fascicularis, rat, and mouseLY6H were maintained in DMEM medium (Invitrogen) with 10% fetal bovineserum (FBS) (Sigma) in the presence of 2 ug/ml puromycin (Invitrogen).

Flow Cytometry

Staining for flow cytometry was performed in 1× cold PBS with 0.5% BSA.Primary antibodies (1 ug/ml) were incubated with live cells on ice for30 minutes. After a brief wash, cells were incubated with Alexa Fluro®488-conjugated anti-human IgG secondary antibody @1:1000 (709-546-149,Jackson ImmunoResearch). Acquisition of the data was performed on aMACSQuant® Flow Cytometers (Miltenyi Biotec) and analyzed with FlowJo™software.

Results

To evaluate the binding of anti-LY6H human monoclonal antibodies to LY6Hin different species. 293 cells overexpressing human, Macacafascicularis, rat, and mouse LY6H were generated. While all antibodieshind human and Macaca fascicularis LY6H (FIG. 3A and FIG. 3B), none ofthem bind efficiently to 293 cells expressing either rat or mouse LY6H(FIG. 3C and FIG. 3D).

Example 5. Estimation of Apparent Antibody Affinity by Flow-Based Assay

Experiments were performed to characterize EC₅₀ of anti-LY6H antibodiesusing the following methods.

Methods

Measurement of Antibody EC₅₀ by Flow Cytometry

The assay uses 3-5 fold dilutions (from 100 nM to 0.1 nM) of antibody ofa stock antibody concentration (100 nM) in binding buffer. Serialdiluted antibodies were incubated with live cells on ice for 30 minutes,after a brief wash, cells were incubated with Alexa Fluro®488-conjugated anti-human IgG secondary antibody @1:100, or 15 ug/ml(709-546-149, Jackson ImmunoResearch). Acquisition of the MFI (MedianFluorescent Intensity) was performed on a MACSQuant® Flow Cytometers(Miltenyi Biotec). EC₅₀ was calculated using Dose-response EC₅₀ shift byglobal fitting from GraphPad Prism.

Results

EC₅₀ or the concentration of antibody that gives half-maximal binding isdetermined by direct and saturable binding of an antibody dilutionseries to both target positive and negative cell lines. An estimate ofaffinity is interpreted from one-half the concentration at whichantibody binding first achieves saturation. EC₅₀ was measured in thefollowing cell lines H446, DMS79, H526, and MOLT3 (FIG. 4). Though EC₅₀may not provide an accurate measure of affinity, it allows for rankingordering of antibodies. 12G7, 11D9, 10B10, and 6D11 were ranked top,followed by 34H6, 1F8, and 2B11.

Example 6. Measurement of Antibody Affinity by Flow Cytometry

Experiments were performed to measure Kd of anti-LY6H antibodies on 293Tcells expressing LY6H. The following methods were used.

Methods

Measurement of Ab Affinity by Flow Cytometry

1 nM PE conjugated antibody was incubated with 2 fold serial dilutions(from 100 nM to 0.05 nM) of unconjugated antibody in binding buffer.Serial diluted antibodies were incubated with live cells on ice for 4hours, after a brief wash. Acquisition of the MFI (Median FluorescentIntensity) was performed on a MACSQuant® Flow Cytometers (MiltenyiBiotec). On cell affinity (Kd) was calculated using Dose-response EC50shift by global fitting from GraphPad Prism™

Results

To measure on cell antibody affinity by flow, 12G7 and IF8 wereconjugated with PE. 293T cells expressing either human Ly6H or Macacafascicularis Ly6H were incubated with PE conjugated antibodies in thepresence of serial dilution of unconjugated antibody. As shown in FIG.5, antibodies bind human and monkey LY6H with similar affinity. Inaddition, cell based affinities are very similar to those measured byOctet™.

Example 7. Internalization of Anti-LY6H Antibody in H446 Cell Line

Experiments were performed to characterize anti-LY6H antibodyinternalization in H446 cells. The following methods were used.

Methods

Tissue Culture and Cell Lines

Human leukemia cell line H446 was obtained from American Type CultureCollection (ATCC). The cells were maintained in RPMI-1640 medium(Invitrogen) with 10% fetal bovine serum (FBS) (Sigma).

Internalization Assay

Live H446 cells were incubated with 12G7 antibody for 30 minutes at 37°C. After cytospin, cells were fixed with 4% PFA and permeabilized with100% methanol, and stained with LAMP1 antibody (#9091, Cell SignalingTechnology, Inc.).

Results

Antibody 12G7 was co-localized to lysozyme, marked by LAMP1 antibody(see FIG. 6).

Example 8. Antibody Internalization and In Vitro Cytotoxicity by 2^(nd)ADC

Experiments were performed to characterize anti-LY6H antibodyinternalization and in vitro cytotoxicity. The following methods wereused.

Methods

Tissue Culture and Cell Lines

Human SCLC cancer cell lines H446, DMS79, NCI-H526, H69 were obtainedfrom American Type Culture Collection (ATCC). Human leukemia cell linesMOLT3, KE-37, EOL1 were purchased from DSMZ. Cells were maintained inRPMI-1640 medium (Invitrogen) with 10% fetal bovine serum (FBS) (Sigma).

Internalization and In Vitro Cytotoxicity

5000 cells/50 ul/well of different cell lines were plated in 96-wellmicroplates. Primary antibodies (0.01 ug/ml, or 0.1 ug/ml finalconcentration) and Fab-Zap or FabFc-Zap @ 0.2 ug/ml final concentration(Advanced Targeting Systems) were added in a volume of 50 ul. The plateswere incubated for 72 hours at 37° C. in the presence of 5% CO2. Foreach plate, 100 ul/well of Cell Titer-Glo™ reagent (# G7573 and # G9243,Promega) was added and allowed to shake for 2 minutes and incubate atroom temperature for 30 minutes prior to reading on a luminescent platereader, and data analyzed using GraphPad Prism™. Transferrin receptor(TR) and hIgG1 antibodies were included as positive and negativecontrols.

Results

Antibody internalization and secondary ADC efficacy was evaluated withFab-Za™p (or FabFc-Zap™) as a conjugated secondary reagent. Fab-ZAP™uses a human primary antibody to target and eliminate cells. Thissecondary conjugate is used to evaluate the potential of a primaryantibody to internalize. Most antibodies showed good efficacy in bothH446 (FIG. 7A) and MOLT3 (FIG. 7B) cell lines, indicating that theseantibodies underwent internalization, released saporin toxin insidecells, and these two cell lines were sensitive to saporin. On the otherhand, there was no cytotoxicity in LY6H negative H69 cell line (FIG.7C).

Example 9. In Vitro Cytotoxicity by Primary Antibody Drug Conjugates

Experiments were performed to characterize anti-LY6H MMAE or PBDconjugated antibody in vitro efficacy. The following methods were used.

Methods

Tissue Culture and Cell Lines

Human SCLC cancer cell lines H446, DMS79, NCI-H526, H69, NCI-H1092, andCORL95 were obtained from American Type Culture Collection (ATCC). Humanleukemia cell lines MOLT3, KE-37, EOL1 were purchased from DSMZ. Cellswere maintained in RPMI-1640 medium (Invitrogen) with 10% fetal bovineserum (FBS) (Sigma).

Antibody Drug Conjugation

Human monoclonal antibodies were conjugated to thevaline-alanine-pyrrolobenzodiazepine, MA-PEGS-VA-PAB-SG3199 (PBD) ormaleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl-monomethylauristatin E (MMAE) as previously described (Stefano J. E., Busch M.,Hou L., Park A., Gianolio D. A. (2013) Micro- and Mid-ScaleMaleimide-Based Conjugation of Cytotoxic Drugs to Antibody Hinge RegionThiols for Tumor Targeting. In: Ducry L. (eds) Antibody-Drug Conjugates.Methods in Molecular Biology (Methods and Protocols), vol 1045. HumanaPress, Totowa, N.J.).

Briefly, each antibody was partially reduced with an appropriate molaramount of TCEP for 2 hours at 37° C. After incubation, the antibodieswere cooled to room temperature and a 6-fold molar excess of PBD or MMAEwas added to the partially reduced antibody for 1 hour. After thisincubation, the entire mixture was run over a gel filtration column toremove free drug. A fraction of the eluate was then used forconcentration and DAR determination using A280 and HIC respectively. Thedrug to antibody ratio for 12G7-MMAE, 1F8-MMAE was 3.14 and 2.0,respectively. The drug to antibody ratio for control human IgG1-vc-MMAE(Ctrl) was 3.2. The drug to antibody ratio for 12G7-PBD and 1F8-PBD was2.1 and 2.5, respectively. The drug to antibody ratio for control humanIgG1-PBD (Ctrl) was 3.0.

In Vitro Efficacy

Cells were seeded onto 96 well plate at 3000-5000 cells/well. ADCs wereadded to the wells in complete culture medium in a serial dilution. Eachtreatment was replicated in 2 wells. 5 days later, cell viability wasmeasured by CellTiter Glo Luminescent Cell™ viability assay (Promega)according to manufacturer's instructions. Cell viability was graphed byPrism™ using ratio of cell viability of test conditions to that ofcontrol wells that are treated with growth medium only.

Results

12G7 and 1F8 were selected to be conjugated to either vcMMAE, amicrotubule inhibitor, or Tesirine PBD, a DNA damage agent. Primaryconjugated antibodies were evaluated for in vitro efficacy against apanel of SCLC and T cell acute lymphoblastic leukemia (T-ALL) celllines.

DMS79, NCI-H1092, and CORL95 are classic SCLC cell lines, whichrepresents 70-80% primary SCLC tumors. H446 and NCI-H526 are consideredto be variant SCLC cell lines, and represent 20-30% primary SCLC tumors.PBD conjugated antibodies showed great efficacy in all SCLC and T-ALLcell lines expressing LY6H, MMAE conjugated antibodies were efficaciousonly in H446 (SCLC) and MOLT3 (T-ALL cell line) (FIG. 8A-8F), suggestingDNA damaging agents, such as PBD are more efficacious than MMAE in avariety of SCLC cell lines. H69 served as a negative control cell line(FIG. 8G).

Example 10. In Vivo Efficacy by Primary Antibody Drug Conjugates

Experiments were performed to characterize in vivo efficacy of drugconjugated antibodies against H446, a variant SCLC cell line. Thefollowing methods were used.

Methods

Tissue Culture and Cell Lines

Human SCLC cancer cell line H446 was obtained from American Type CultureCollection (ATCC). Cells were maintained in RPMI-1640 medium(Invitrogen) with 10% fetal bovine serum (FBS) (Sigma).

Antibody Drug Conjugation

Human monoclonal antibodies were conjugated to thevaline-alanine-pyrrolobenzodiazepine, MA-PEGS-VA-PAB-SG3199 (PBD) ormaleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl-monomethylauristatin E (MMAE) as previously described (Stefano J. E., Busch M.,Hou L., Park A., Gianolio D. A. (2013) Micro- and Mid-ScaleMaleimide-Based Conjugation of Cytotoxic Drugs to Antibody Hinge RegionThiols for Tumor Targeting. In: Ducry L. (eds) Antibody-Drug Conjugates.Methods in Molecular Biology (Methods and Protocols), vol 1045. HumanaPress, Totowa, N.J.) Briefly, each antibody was partially reduced withan appropriate molar amount of TCEP for 2 hours at 37° C. Afterincubation, the antibodies were cooled to room temperature and a 6-foldmolar excess of PBD or MMAE was added to the partially reduced antibodyfor 1 hour. After this incubation, the entire mixture was run over a gelfiltration column to remove free drug. A fraction of the eluate was thenused for concentration and DAR determination using A280 and HICrespectively. The drug to antibody ratio for 12G7-MMAE was 3.14. Thedrug to antibody ratio for control human IgG1-vc-MMAE (Ctrl) was 3.2.The drug to antibody ratio for 12G7-PBD and 1F8-PBD was 2.1 and 2.5,respectively. The drug to antibody ratio for control human IgG1-PBD(Ctrl) was 3.0.

Tumor Implantation

For efficacy studies with antibody drug conjugates, 6-8 weeks old femaleNu/Nu mice obtained from Taconic were inoculated subcutaneously in thedorsal right flank with 2.5 million H446 cells in 1×PBS with equalvolume of Matrigel™ (#356234, Corning). When tumor volumes reached150-200 mm3 (day 0), animals were randomized into 3 groups of 6-7 eachand administered BIW×2 IV injection of antibody drug conjugates. Tumorvolumes were determined using digital calipers (Fred V. Fowler Company,Inc.) using the formula (length×width²)/2. Measurement was performedtwice weekly for the first 30 days after initial dosing, then once aweek until the end of the study.

Toxicity

Animals were weighed weekly for the study. The mice were observedfrequently for overt signs of any adverse, treatment-related sideeffects, and clinical signs of toxicity were recorded when observed.Acceptable toxicity is defined as a group mean body-weight loss of lessthan 20% during the study and not more than one treatment-related (TR)death among ten treated animals. Any dosing regimen that results ingreater toxicity is considered above the maximum tolerated dose (MTD). Adeath is classified as TR if attributable to treatment side effects asevidenced by clinical signs and/or necropsy or if due to unknown causesduring the dosing period or within 10 days of the last dose. A death isclassified as an NTR if there is no evidence that death was related totreatment side effects.

Results

MMAE conjugated antibodies were dosed at 5 mg/kg 4 times in 2 weeks. The12G7-MMAE treated group showed tumor growth inhibition and survivaladvantage over the control groups (FIG. 9). Meanwhile, Tesirine PBDconjugated antibodies dosed at 1 mg/kg 3 times every 4 days showed tumorregression, excellent efficacy and survival advantage over the controlgroups (FIG. 10).

Example 11. In Vivo Efficacy by Primary Antibody Drug Conjugates

Experiments were performed to characterize in vivo efficacy of drugconjugated antibodies against H526, a variant SCLC cell line. Thefollowing methods were used.

Methods

Tissue Culture and Cell Lines

Human SCLC cancer cell line H526 was obtained from American Type CultureCollection (ATCC). Cells were maintained in RPMI-1640 medium(Invitrogen) with 10% fetal bovine serum (FBS) (Sigma).

Antibody Drug Conjugation

Human monoclonal antibodies were conjugated to thevaline-alanine-pyrrolobenzodiazepine, MA-PEGS-VA-PAB-SG3199 (PBD) ormaleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl-monomethylauristatin E (MMAE) as previously described (Stefano J. E., Busch M.,Hou L., Park A., Gianolio D. A. (2013) Micro- and Mid-ScaleMaleimide-Based Conjugation of Cytotoxic Drugs to Antibody Hinge RegionThiols for Tumor Targeting. In: Ducry L. (eds) Antibody-Drug Conjugates.Methods in Molecular Biology (Methods and Protocols), vol 1045. HumanaPress, Totowa, N.J.) Briefly, each antibody was partially reduced withan appropriate molar amount of TCEP for 2 hours at 37° C. Afterincubation, the antibodies were cooled to room temperature and a 6-foldmolar excess of PBD or MMAE was added to the partially reduced antibodyfor 1 hour. After this incubation, the entire mixture was run over a gelfiltration column to remove free drug. A fraction of the eluate was thenused for concentration and DAR determination using A280 and HICrespectively. The drug to antibody ratio for 12G7-MMAE was 3.14. Thedrug to antibody ratio for control human IgG1-vc-MMAE (Ctrl) was 3.2.The drug to antibody ratio for 12G7-PBD and 1F8-PBD was 2.1 and 2.5,respectively. The drug to antibody ratio for control human IgG1-PBD(Ctrl) was 3.0.

Tumor Implantation

For efficacy studies with antibody drug conjugates, 6-8 weeks old femaleNu/Nu mice from Taconic were inoculated subcutaneously in the dorsalright flank with 2.5 million H526 cells in 1×PBS with equal volume ofMatrigel (#356234, Corning). When tumor volumes reached 150-200 mm3 (day0), animals were randomized into 3 groups of 6-7 each and administeredBIW×2 IV injection of antibody drug conjugates. Tumor volumes weredetermined using digital calipers (Fred V. Fowler Company, Inc.) usingthe formula (length×width²)/2. Measurement was performed twice weeklyfor the first 30 days after initial dosing, then once a week till theend of the study.

Toxicity

Animals were weighed weekly for the study. The mice were observedfrequently for overt signs of any adverse, treatment-related sideeffects, and clinical signs of toxicity were recorded when observed.Acceptable toxicity is defined as a group mean body-weight loss of lessthan 20% during the study and not more than one treatment-related (TR)death among ten treated animals. Any dosing regimen that results ingreater toxicity is considered above the maximum tolerated dose (MTD). Adeath is classified as TR if attributable to treatment side effects asevidenced by clinical signs and/or necropsy or if due to unknown causesduring the dosing period or within 10 days of the last dose. A death isclassified as an NTR if there is no evidence that death was related totreatment side effects.

Results

MMAE conjugated antibodies were dosed at 5 mg/kg 4 times in 2 weeks. The12G7-MMAE treated group showed some tumor growth inhibition and survivaladvantage over the control groups (FIG. 11). On the other hand, TesirinePBD conjugated antibodies dosed at 1 mg/kg 3 times every 4 days showedtumor regression, excellent efficacy and survival advantage over thecontrol groups (FIG. 12). Little or no mice body weight loss wasobserved for the study (FIGS. 11C and 12C).

Example 12. In Vivo Efficacy by Single Dose Primary Antibody DrugConjugates

Experiments were performed to characterize in vivo efficacy of drugconjugated antibodies against DMS79, a classic SCLC cell line. Thefollowing methods were used.

Methods

Tissue Culture and Cell Lines

Human SCLC cancer cell line DMS79 was obtained from American TypeCulture Collection (ATCC). Cells were maintained in RPMI-1640 medium(Invitrogen) with 10% fetal bovine serum (FBS) (Sigma).

Antibody Drug Conjugation

Human monoclonal antibodies were conjugated to thevaline-alanine-pyrrolobenzodiazepine, MA-PEGS-VA-PAB-SG3199 (PBD aspreviously described (Stefano J. E., Busch M., Hou L., Park A., GianolioD. A. (2013) Micro- and Mid-Scale Maleimide-Based Conjugation ofCytotoxic Drugs to Antibody Hinge Region Thiols for Tumor Targeting. In:Ducry L. (eds) Antibody-Drug Conjugates. Methods in Molecular Biology(Methods and Protocols), vol 1045. Humana Press, Totowa, N.J.). Briefly,each antibody was partially reduced with an appropriate molar amount ofTCEP for 2 hours at 37° C. After incubation, the antibodies were cooledto room temperature and a 6-fold molar excess of PBD was added to thepartially reduced antibody for 1 hour. After this incubation, the entiremixture was run over a gel filtration column to remove free drug. Afraction of the eluate was then used for concentration and DARdetermination using A280 and HIC respectively. The drug to antibodyratio for 12G7-PBD was 2.0. The drug to antibody ratio for control humanIgG1-PBD (Ctrl) was 2.9.

Tumor Implantation

For efficacy studies with antibody drug conjugates, 6-8 weeks old femaleNu/Nu mice from Taconic were inoculated subcutaneously in the dorsalright flank with 2.5 million DMS79 cells in 1×PBS with equal volume ofMatrigel (#356234, Corning). When tumor volumes reached 150-200 mm3 (day0), animals were randomized into 3 groups of 7-8 each and administeredonce IV injection of antibody drug conjugates. Tumor volumes weredetermined using digital calipers (Fred V. Fowler Company, Inc.) usingthe formula (length×width²)/2. Measurement was performed twice weeklyfor the first 30 days after initial dosing, then once a week till theend of the study.

Toxicity

Animals were weighed weekly for the study. The mice were observedfrequently for overt signs of any adverse, treatment-related sideeffects, and clinical signs of toxicity were recorded when observed.Acceptable toxicity is defined as a group mean body-weight loss of lessthan 20% during the study and not more than one treatment-related (TR)death among ten treated animals. Any dosing regimen that results ingreater toxicity is considered above the maximum tolerated dose (MTD). Adeath is classified as TR if attributable to treatment side effects asevidenced by clinical signs and/or necropsy or if due to unknown causesduring the dosing period or within 10 days of the last dose. A death isclassified as an NTR if there is no evidence that death was related totreatment side effects.

Results

DMS79 is a classic SCLC cell line, which refers to the usual recognizedform of SCLC, with typical morphology, expression of neuroendocrine (NE)properties and a non-adherent growth pattern in vitro. Tesirine PBDconjugated antibody (12G7-PBD) was dosed at 1 mg/kg once, and showedtumor regression, excellent efficacy and survival advantage over thecontrol groups (FIG. 13), with no loss of body weight.

Example 13. In Vivo Efficacy by Single Dose Primary Antibody DrugConjugates

Experiments were performed to characterize in vivo efficacy of drugconjugated antibodies against H446, a variant SCLC cell line. Thefollowing methods were used.

Methods

Tissue Culture and Cell Lines

Human SCLC cancer cell line H446 was obtained from American Type CultureCollection (ATCC). Cells were maintained in RPMI-1640 medium(Invitrogen) with 10% fetal bovine serum (FBS) (Sigma).

Antibody drug conjugation Human monoclonal antibodies were conjugated tothe valine-alanine-pyrrolobenzodiazepine, MA-PEGS-VA-PAB-SG3199 (PBD aspreviously described (Stefano J. E., Busch M., Hou L., Park A., GianolioD. A. (2013) Micro- and Mid-Scale Maleimide-Based Conjugation ofCytotoxic Drugs to Antibody Hinge Region Thiols for Tumor Targeting. In:Ducry L. (eds) Antibody-Drug Conjugates. Methods in Molecular Biology(Methods and Protocols), vol 1045. Humana Press, Totowa, N.J.) Briefly,each antibody was partially reduced with an appropriate molar amount ofTCEP for 2 hours at 37° C. After incubation, the antibodies were cooledto room temperature and a 6-fold molar excess of PBD was added to thepartially reduced antibody for 1 hour. After this incubation, the entiremixture was run over a gel filtration column to remove free drug. Afraction of the eluate was then used for concentration and DARdetermination using A280 and HIC respectively. The drug to antibodyratio for 12G7-PBD and 1F8-PBD was 2.3 and 2.2, respectively. The drugto antibody ratio for control human IgG1-PBD (Ctrl) was 2.1.

Tumor Implantation

For efficacy studies with antibody drug conjugates, 6-8 weeks old femaleNu/Nu mice from Taconic were inoculated subcutaneously in the dorsalright flank with 2.5 million H446 cells in 1×PBS with equal volume ofMatrigel (#356234, Corning). When tumor volumes reached 150-200 mm3 (day0), animals were randomized into 3 groups of 7-8 each and administeredonce IV injection of antibody drug conjugates. Tumor volumes weredetermined using digital calipers (Fred V. Fowler Company, Inc.) usingthe formula (length×width²)/2. Measurement was performed twice weeklyfor the first 30 days after initial dosing, then once a week till theend of the study.

Toxicity

Animals were weighed weekly for the study. The mice were observedfrequently for overt signs of any adverse, treatment-related sideeffects, and clinical signs of toxicity were recorded when observed.Acceptable toxicity is defined as a group mean body-weight loss of lessthan 20% during the study and not more than one treatment-related (TR)death among ten treated animals. Any dosing regimen that results ingreater toxicity is considered above the maximum tolerated dose (MTD). Adeath is classified as TR if attributable to treatment side effects asevidenced by clinical signs and/or necropsy or if due to unknown causesduring the dosing period or within 10 days of the last dose. A death isclassified as an NTR if there is no evidence that death was related totreatment side effects.

Results

H446 is a variant subtype of SCLC cell line, characterized by largercells with prominent nucleoli, partial or complete loss ofneuroendocrine (NE) cell properties, partial adherent growth in vitro,frequent MYC amplification and epithelial-mesenchymal transition.

Tesirine PBD conjugated antibodies were dosed at 0.1, 0.3, and 1 mg/kgonce. While there was little efficacy when mice were dosed at 0.1 and0.3 mg/kg, when mice were dosed at 1 mg/kg, tumor growth inhibition andsurvival advantage over the control groups was shown, especially with1F8 PBD conjugated antibody (FIG. 14). There was no loss of mice bodyweight during the study.

Example 14: In Vitro Efficacy of an Anti-LY6H-PBD Antibody Combined withOlaparib, Cisplatin, or Etoposide

Cisplatin and etoposide have been used as first line treatment for SCLCpatients. Thus, a combination of an anti-LY6H antibody conjugated to PBD(12G7-PBD) with cisplatin or etoposide was tested. Furthermore, Poly[ADP-ribose] polymerase 1 (PARP1) was identified to be highly expressedat the mRNA and protein level in SCLC (Byers, L. A., et al, ProteomicProfiling Identifies Dysregulated Pathways in Small Cell Lung Cancer andNovel Therapeutic Targets Including PARP1, Cancer Discov., 2012). Thus,PARP inhibitor and olaparib were combined, with 12G7-PBD to determinethe effect on growth in three classic SCLC cell lines (DMS79, CORL95,and NCI-H1092) in vitro.

Methods

SCLC cells were seeded onto 96 well plates at 3000-5,000 cells/well onthe day of treatment. To distinguish synergy from additivity of thecombination of LY6H-PBD with Olaparib (selleckchem, Cat # S1060),etoposide (Cell Signaling Technology, Cat #2200S) or cisplatin (Sigma,Cat # P4394), for each combination, a specific isobologram wasconstructed. IC50 value was defined as the drug concentration reducingby 50% the growth of treated cells compared with control. IC50 values ofeach drug was determined by plating cells and adding 12G7-PBD, olaparib,cisplatin, or etoposide at 10-fold dilutions spanning their probablerange of activity. Concentrations ranging from 0.0001-10 ug/ml were usedfor 12G7-PBD, from 0.0001 to 100 uM for olaparib, cisplatin, andetoposide. Cell viability was measured 5 days later by CellTiter Glo®Luminescent Cell viability Assay™ (Promega, Madison, Wis.) according tomanufacturer's instructions. Each IC50 was graphically derived from thegrowth curve graphed by Prism™ showing percent survival vs log drugconcentrations.

The 50% growth-inhibitory activity of the drug combinations was measuredafter 5 days of treatment. The line of additivity for the isobologramwas constructed by interpolating the two points corresponding to theIC50 of the two drugs alone. Graphically, synergy, additivity, andantagonism are indicated by a point plotted below, on, or above the lineof additivity, respectively.

Results

The IC50 value of 12G7-PBD, olaparib, cisplatin, and etoposide alone wasdetermined in DMS79 cell line. The IC50 of 12G7-PBD was 25.12 pM inDMS79 cell line. The IC50 of olaparib was 4.07 uM. The IC50 of cisplatinwas 0.31 uM. The IC50 of etoposide was 0.29 uM. An isobologram analysiswas performed of the combination of 12G7-PBD at 7.9 pM, or 18.8 pM withtitrating concentrations of olaparib, cisplatin, or etoposide (see FIG.15A-15C). It was then assessed if the combination results in additive orsynergistic effect based on the isobologram.

When 12G7-PBD was combined with olaparib, cisplatin, or etoposide,synergistic effect in DMS79 cells was observed (FIG. 15). Similarsynergistic effects were observed in CORL95 cells (FIG. 16). Inaddition, synergistic effect was observed in NCI-H1092 cells when12G7-PBD was combined with either olaparib or etoposide (FIGS. 17A,17C). Additive effect was observed when 12G7-PBD was combined withcisplatin (FIG. 17B).

Sequence Summary

SEQ ID NO: Description 1 6D11 VH amino acid sequence 2 6D11, 6D11_41B4VH CDR1 amino acid sequence 3 6D11 VH CDR2 amino acid sequence 4 6D11,6D11_41B4 VH CDR3 amino acid sequence 5 6D11, 6D11_41B4 VL amino acidsequence 6 6D11, 12G7, 10B10, 12G7_S54A, 12G7_N52Q, 6D11_41B4,10B10_S54A VL CDR1 amino acid sequence 7 6D11, 12G7, 10B10, 12G7_S54A,12G7_N52Q, 6D11_41B4, 10B10_S54A VL CDR2 amino acid sequence 8 6D11,12G7, 10B10, 12G7_S54A, 12G7_N52Q, 6D11_41B4, 10B10_S54A VL CDR3 aminoacid sequence 9 11D9 VH amino acid sequence 10 11D9 VH CDR1 amino acidsequence 11 11D9, 23F3 VH CDR2 amino acid sequence 12 11D9 VH CDR3 aminoacid sequence 13 11D9 VL amino acid sequence 14 11D9, 23F3 VL CDR1 aminoacid sequence 15 11D9, 23F3 VL CDR2 amino acid sequence 16 11D9 VL CDR3amino acid sequence 17 23F3 VH amino acid sequence 18 23F3 VH CDR1 aminoacid sequence 19 23F3 VH CDR3 amino acid sequence 20 23F3 VL amino acidsequence 21 23F3 VL CDR3 amino acid sequence 22 12G7 VH amino acidsequence 23 12G7, 12G7_S54A,, 12G7_N52Q VH CDR1 amino acid sequence 2412G7 VH CDR2 amino acid sequence 25 12G7, 12G7_S54A, 12G7_N52Q VH CDR3amino acid sequence 26 12G7, 10B10, 12G7_S54A, 12G7_N52Q, 10B10_S54A VLamino acid sequence 27 22A5 VH amino acid sequence 28 22A5, 26F3, 35H10,24A10, 34H6 VH CDR1 amino acid sequence 29 22A5, 26F3, 35H10, 2B11,24A10, 34H6 VH CDR2 amino acid sequence 30 22A5 VH CDR3 amino acidsequence 31 22A5 VL amino acid sequence 32 22A5, 26F3 VL CDR1 amino acidsequence 33 22A5, 26F3 VL CDR2 amino acid sequence 34 22A5 VL CDR3 aminoacid sequence 35 26F3 VH amino acid sequence 36 26F3 VH CDR3 amino acidsequence 37 26F3 VL amino acid sequence 38 26F3 VL CDR3 amino acidsequence 39 35H10 VH amino acid sequence 40 35H10 VH CDR3 amino acidsequence 41 35H10 VL amino acid sequence 42 35H10 VL CDR1 amino acidsequence 43 35H10 VL CDR2 amino acid sequence 44 35H10 VL CDR3 aminoacid sequence 45 1F8 VH amino acid sequence 46 1F8, 2B11 VH CDR1 aminoacid sequence 47 1F8 VH CDR2 amino acid sequence 48 1F8, 2B11 VH CDR3amino acid sequence 49 1F8, 2B11 VL amino acid sequence 50 1F8, 2B11,24A10, 34H6 VL CDR1 amino acid sequence 51 1F8, 2B11, 24A10, 34H6 VLCDR2 amino acid sequence 52 1F8, 2B11 VL CDR3 amino acid sequence 532B11 VH amino acid sequence 54 24A10 VH amino acid sequence 55 24A10 VHCDR3 amino acid sequence 56 24A10, 34H6, 34H6 VL amino acid sequence 5724A10, 34H6, 34H6 VL CDR3 amino acid sequence 58 34H6 VH amino acidsequence 59 34H6 VH CDR3 amino acid sequence 60 10B10 VH amino acidsequence 61 10B10, 10B10_S54A VH CDR1 amino acid sequence 62 10B10 VHCDR2 amino acid sequence 63 10B10, 10B10_S54A VH CDR3 amino acidsequence 64 12G7_S54A VH amino acid sequence 65 12G7_S54A VH CDR2 aminoacid sequence 66 12G7_N52Q VH amino acid sequence 67 12G7_N52Q VH CDR2amino acid sequence 68 6D11_41B4 VH amino acid sequence 69 6D11_41B4 VHCDR2 amino acid sequence 70 10B10_S54A VH amino acid sequence 7110B10_S54A CDR2 amino acid sequence 72 6D11 VH DNA sequence 73 6D11,6D11_41B4 VL DNA sequence 74 11D9 VH DNA sequence 75 11D9 VL DNAsequence 76 23F3 VH DNA sequence 77 23F3 VL DNA sequence 78 12G7 VH DNAsequence 79 12G7, 10B10, 12G7_S54A, 12G7_N52Q, 10B10_S54A VL DNAsequence 80 22A5 VH DNA sequence 81 22A5 VL DNA sequence 82 26F3 VH DNAsequence 83 26F3 VL DNA sequence 84 35H10 VH DNA sequence 85 35H10 VLDNA sequence 86 1F8 VH DNA sequence 87 1F8, 2B11 VL DNA sequence 88 2B11VH DNA sequence 89 24A10 VH DNA sequence 90 24A10, 34H6 VL DNA sequence91 34H6 VH DNA sequence 92 10B10 VH DNA sequence 93 12G7_S54A VH DNAsequence 94 12G7_N52O VH DNA sequence 95 6D11_41B4 VH DNA sequence 9610B10_S54A VH DNA sequence 97 IGHV4 leader 98 IGHV2 leader 99 IGHV2-26leader 100 IGHV6 leader 101 IGHV1 leader 102 IGHV1-58 leader 103IGHV1-24 leader 104 IGHV1-69/1-46/7-4-1 leader 105 IGHV3 leader 106IGHV3-53/3-49 leader 107 IGHV3-21 leader 108 IGHV3-48/3-7 leader 109IGHV5 leader 110 IgkV1a leader 111 IgkV1b leader 112 IgkV3 leader 113IgkV3-20 leader 114 IgkV4 leader 115 IgkV5 leader 116 IgkV2 leader 117Kappa FW4 118 Kappa FW4 119 Heavy FW4 120 VL-FOR L1 121 VL-FOR L2 122VL-REV L 123 LY6H_HUMAN 124 LY6H_MACFA 125 NP_001128311.1 126 LY6H_MOUSE

INCORPORATION BY REFERENCE

The contents of all references, patents, pending patent applications andpublished patents, Sequence Listing, Sequence Summary, and AccessionNumbers, cited throughout this application are hereby expresslyincorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1.-42. (canceled)
 43. An anti-LY6H antibody, or antigen-binding portionthereof, comprising a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 25, a CDR2domain comprising one of the amino acid sequences selected from SEQ IDNO: 24, SEQ ID NO: 65 and SEQ ID NO: 67, and a CDR1 domain comprisingthe amino acid sequence of SEQ ID NO: 23, and a light chain variableregion comprising a CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of SEQ IDNO: 7, and a CDR1 domain comprising the amino acid sequence of SEQ IDNO: 6; or a heavy chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 48, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 47, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 46; and a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 52, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 51, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO:
 50. 44.-58. (canceled)
 59. The anti-LY6Hantibody, or antigen-binding portion thereof, of claim 43, wherein theheavy chain variable region comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 22, SEQ ID NO: 64, and SEQ IDNO: 66 and the light chain variable region comprises the amino acidsequence of SEQ ID NO: 26; or wherein the heavy chain variable regioncomprises an amino acid sequence of SEQ ID NO: 45 and the light chainvariable region comprises the amino acid sequence of SEQ ID NO:
 49. 60.An anti-LY6H antibody, or antigen-binding portion thereof, comprising aheavy chain comprising an amino acid sequence set forth in one of SEQ IDNO: 22, SEQ ID NO: 64 and SEQ ID NO: 66, or a sequence having at least90%, 95%, 96%, 97%, 98%, or 99% identity to one of SEQ ID NO: 22, SEQ IDNO: 64 and SEQ ID NO: 66, and/or a light chain comprising an amino acidsequence set forth in SEQ ID NO: 26, or a sequence having at least 90%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26; or a heavy chainvariable region comprising an amino acid sequence having at least 90%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 45 and a light chainvariable region comprising an amino acid sequence having at least 90%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:
 49. 61.-74. (canceled)75. An antibody, or antigen-binding portion thereof, that binds to thesame epitope as the antibody, or antigen-binding portion thereof, ofclaim
 43. 76. An isolated nucleic acid encoding an antibody, or antigenbinding portion thereof, of any one claim
 43. 77. A pharmaceuticalcomposition comprising the antibody, or antigen binding portion thereof,of claim 43, and a pharmaceutically acceptable carrier.
 78. An antibody,or antigen binding portion thereof, of claim 43, conjugated to at leastone drug. 79.-121. (canceled)
 122. The antibody, or antigen bindingportion thereof, of claim 43, wherein the antibody, or antigen bindingportion thereof, is an IgG1 isotype. 123.-124. (canceled)
 125. A methodof treating cancer in a subject, the method comprising administering atherapeutically effective amount of the antibody, or antigen bindingportion thereof, of claim 43, to the subject, thereby treating thecancer.
 126. The method of claim 125, wherein the cancer is small celllung cancer.
 127. The method of claim 126, wherein the small cell lungcancer is a classic small cell lung cancer or a variant small cell lungcancer.
 128. A method of inhibiting or decreasing tumor growth in asubject, the method comprising administering a therapeutically effectiveamount of the antibody, or antigen binding portion thereof, of claim 43to the subject, thereby inhibiting or decreasing tumor growth in thesubject. 129.-130. (canceled)
 131. The method of claim 125, wherein theantibody, or antigen binding portion thereof, is administered incombination with an additional agent or an additional therapy.
 132. Themethod of claim 131, wherein the additional agent is an immunecheckpoint inhibitor.
 133. The method of claim 132, wherein the immunecheckpoint inhibitor is an antibody.
 134. The method of claim 133,wherein the antibody is selected from the group consisting of ananti-PD1 antibody, an anti-PD-L1 antibody or an anti-CTLA-4 antibody.135.-136. (canceled)
 137. The method of claim 125, wherein the cancer ortumor is characterized as having LY6H expression or overexpression.138.-141. (canceled)